Science.gov

Sample records for embryonic globin genes

  1. Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

    PubMed

    Singal, Rakesh; vanWert, Jane M; Ferdinand, Larry

    2002-12-01

    The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex. PMID:12393573

  2. Differential loss of embryonic globin genes during the radiation of placental mammals

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Storz, Jay F.

    2008-01-01

    The differential gain and loss of genes from homologous gene families represents an important source of functional variation among the genomes of different species. Differences in gene content between species are primarily attributable to lineage-specific gene gains via duplication and lineage-specific losses via deletion or inactivation. Here, we use a comparative genomic approach to investigate this process of gene turnover in the β-globin gene family of placental mammals. By analyzing genomic sequence data from representatives of each of the main superordinal clades of placental mammals, we were able to reconstruct pathways of gene family evolution during the basal radiation of this physiologically and morphologically diverse vertebrate group. Our analysis revealed that an initial expansion of the nonadult portion of the β-globin gene cluster in the ancestor of placental mammals was followed by the differential loss and retention of ancestral gene lineages, thereby generating variation in the complement of embryonic globin genes among contemporary species. The sorting of ε-, γ-, and η-globin gene lineages among the basal clades of placental mammals has produced species differences in the functional types of hemoglobin isoforms that can be synthesized during the course of embryonic development. PMID:18755893

  3. Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes

    PubMed Central

    Navas, Patrick A.; Swank, Richard A.; Yu, Man; Peterson, Kenneth R.; Stamatoyannopoulos, George

    2010-01-01

    High-level β-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1–HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb β-globin locus yeast artificial chromosome and measuring the activity of β-globin genes in GT6m β-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the β-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of ε- and γ-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, γ-globin gene expression was significantly reduced while β-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal ε- and γ-globin gene expression during embryonic erythropoiesis and for γ-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression. PMID:14506128

  4. Tissue specific transcription of the human epsilon-globin gene following transfection into the embryonic erythroid cell line K562.

    PubMed Central

    Allan, M; Montague, P; Grindlay, G J; Sibbet, G; Donovan-Peluso, M; Bank, A; Paul, J

    1985-01-01

    We have introduced a plasmid containing the human epsilon-globin gene either stably or transiently into a number of erythroid or non-erythroid cell lines, and analysed the accuracy and efficiency of transcription. In non-erythroid cells (or in mouse erythroleukaemia (MEL) cells in which adult but not embryonic globin genes are expressed) transcription of the epsilon-globin gene occurs mainly from a site 200 bp upstream of the major cap site (the -200 cap site). In the human K562 cell line, in which the endogenous epsilon-globin gene is transcribed at high levels, transcription initiation from the introduced gene occurs mainly from the major cap site. Transcriptional activity of the epsilon-globin gene introduced into K562 cell is quantitatively similar to that of the endogenous gene. This suggests the presence (or absence) in K562 cells of factor(s) which activate (or repress) the epsilon-globin gene in a tissue specific manner. Images PMID:2995916

  5. Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements.

    PubMed Central

    Trepicchio, W L; Dyer, M A; Baron, M H

    1993-01-01

    The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene. Images PMID:8246963

  6. Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes.

    PubMed

    Perez-Stable, C; Costantini, F

    1990-03-01

    The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin

  7. Generation of Mice Deficient in both KLF3/BKLF and KLF8 Reveals a Genetic Interaction and a Role for These Factors in Embryonic Globin Gene Silencing

    PubMed Central

    Funnell, Alister P. W.; Mak, Ka Sin; Twine, Natalie A.; Pelka, Gregory J.; Norton, Laura J.; Radziewic, Tania; Power, Melinda; Wilkins, Marc R.; Bell-Anderson, Kim S.; Fraser, Stuart T.; Perkins, Andrew C.; Tam, Patrick P.; Pearson, Richard C. M.

    2013-01-01

    Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development. PMID:23716600

  8. Globin gene switching in primates.

    PubMed

    Johnson, Robert M; Gumucio, Deborah; Goodman, Morris

    2002-11-01

    Evolutionary approaches to the identification of DNA sequences required for transcription of the genes of the beta-globin cluster are reviewed. Sequence alignments of non-coding regions from widely divergent species revealed many conserved motifs (phylogenetic footprints) that were putative transcription factor binding sites and candidate binding proteins were identified. The differential timing of the prosimian and simian gamma-globin genes was analyzed by identifying base changes in the vicinity of the phylogenetic footprints. These differential phylogenetic footprints were shown to bind different nuclear factors, and the behavior of constructs with human or galago gamma-promoters in transgenic mice indicated that DNA motifs near the gamma-globin genes are sufficient to determine the developmental stage of expression. Locus control region alignments have identified many conserved sequence differences outside of the hypersensitive sites. Globin protein and mRNA expression profiles during embryological development in a series of catarrhine (Old World monkeys and apes) and platyrrhine (New World monkeys) primates have been determined. While all catarrhines examined to date have globin expression patterns that are highly similar to the well-established human switching behavior, platyrrhines have inactivated their gamma 1 genes by a variety of mechanisms, and have an earlier gamma-beta switch. PMID:12443943

  9. Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

    PubMed Central

    Patel, Vidushi S; Cooper, Steven JB; Deakin, Janine E; Fulton, Bob; Graves, Tina; Warren, Wesley C; Wilson, Richard K; Graves, Jennifer AM

    2008-01-01

    Background Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation. Results The platypus α-globin cluster (chromosome 21) contains embryonic and adult α- globin genes, a β-like ω-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-ζ-ζ'-αD-α3-α2-α1-ω-GBY-3'. The platypus β-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-ε-β-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate α-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal β-globin clusters are embedded in olfactory genes. Thus, the mammalian α- and β-globin clusters are orthologous to the bird α- and β-globin clusters respectively. Conclusion We propose that α- and β-globin clusters evolved from an ancient MPG-C16orf35-α-β-GBY-LUC7L arrangement 410 million years ago. A copy of the original β (represented by ω in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago), then duplicated and diverged to form orthologous clusters of β-globin genes with different expression profiles in different lineages. PMID:18657265

  10. An embryonic stage–specific enhancer within the murine β-globin locus mediates domain-wide histone hyperacetylation

    PubMed Central

    Fromm, George; Cadiz-Rivera, Brenda; de Vries, Christina; Getman, Michael; McGrath, Kathleen E.; Kingsley, Paul D.; Fields, Jennifer; Fiering, Steven

    2011-01-01

    In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian β-globin gene loci, and determined that within the murine locus, neither the β-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic β-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ϵ-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine β-globin locus results in significant decrease in the expression of the embryonic β-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for β-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure. PMID:21321362

  11. Genomic evidence for independent origins of β-like globin genes in monotremes and therian mammals

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Storz, Jay F.

    2008-01-01

    Phylogenetic reconstructions of the β-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult β-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic β-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the “ε-globin” gene in birds is not orthologous to the ε-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto β-globin gene. Here, we report evidence that the early and late expressed β-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto β-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the ε- and β-globin genes of therian mammals arose via duplication of a proto β-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of β-like globin genes that originated via duplication of a proto β-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto β-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development. PMID:18216242

  12. Transcriptional interference among the murine β-like globin genes

    PubMed Central

    Hu, Xiao; Eszterhas, Susan; Pallazzi, Nicolas; Bouhassira, Eric E.; Fields, Jennifer; Tanabe, Osamu; Gerber, Scott A.; Bulger, Michael; Engel, James Douglas; Groudine, Mark

    2007-01-01

    Mammalian β-globin loci contain multiple genes that are activated at different developmental stages. Studies have suggested that the transcription of one gene in a locus can influence the expression of the other locus genes. The prevalent model to explain this transcriptional interference is that all potentially active genes compete for locus control region (LCR) activity. To investigate the influence of transcription by the murine embryonic genes on transcription of the other β-like genes, we generated mice with deletions of the promoter regions of Ey and βh1 and measured transcription of the remaining genes. Deletion of the Ey and βh1 promoters increased transcription of βmajor and βminor 2-fold to 3-fold during primitive erythropoiesis. Deletion of Ey did not affect βh1 nor did deletion of βh1 affect Ey, but Ey deletion uniquely activated transcription from βh0, a β-like globin gene immediately downstream of Ey. Protein analysis showed that βh0 encodes a translatable β-like globin protein that can pair with alpha globin. The lack of transcriptional interference between Ey and βh1 and the gene-specific repression of βh0 did not support LCR competition among the embryonic genes and suggested that direct transcriptional interference from Ey suppressed βh0. PMID:17077320

  13. Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Natarajan, Chandrasekhar; Witt, Christopher C.; Berenbrink, Michael; Storz, Jay F.

    2015-01-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa. PMID:25502940

  14. Globin gene structure in a reptile supports the transpositional model for amniote α- and β-globin gene evolution.

    PubMed

    Patel, Vidushi S; Ezaz, Tariq; Deakin, Janine E; Graves, Jennifer A Marshall

    2010-12-01

    The haemoglobin protein, required for oxygen transportation in the body, is encoded by α- and β-globin genes that are arranged in clusters. The transpositional model for the evolution of distinct α-globin and β-globin clusters in amniotes is much simpler than the previously proposed whole genome duplication model. According to this model, all jawed vertebrates share one ancient region containing α- and β-globin genes and several flanking genes in the order MPG-C16orf35-(α-β)-GBY-LUC7L that has been conserved for more than 410 million years, whereas amniotes evolved a distinct β-globin cluster by insertion of a transposed β-globin gene from this ancient region into a cluster of olfactory receptors flanked by CCKBR and RRM1. It could not be determined whether this organisation is conserved in all amniotes because of the paucity of information from non-avian reptiles. To fill in this gap, we examined globin gene organisation in a squamate reptile, the Australian bearded dragon lizard, Pogona vitticeps (Agamidae). We report here that the α-globin cluster (HBK, HBA) is flanked by C16orf35 and GBY and is located on a pair of microchromosomes, whereas the β-globin cluster is flanked by RRM1 on the 3' end and is located on the long arm of chromosome 3. However, the CCKBR gene that flanks the β-globin cluster on the 5' end in other amniotes is located on the short arm of chromosome 5 in P. vitticeps, indicating that a chromosomal break between the β-globin cluster and CCKBR occurred at least in the agamid lineage. Our data from a reptile species provide further evidence to support the transpositional model for the evolution of β-globin gene cluster in amniotes. PMID:21116705

  15. Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters

    PubMed Central

    2010-01-01

    Background The vertebrate globin genes encoding the α- and β-subunits of the tetrameric hemoglobins are clustered at two unlinked loci. The highly conserved linear order of the genes flanking the hemoglobins provides a strong anchor for inferring common ancestry of the globin clusters. In fish, the number of α-β-linked globin genes varies considerably between different sublineages and seems to be related to prevailing physico-chemical conditions. Draft sequences of the Atlantic cod genome enabled us to determine the genomic organization of the globin repertoire in this marine species that copes with fluctuating environments of the temperate and Arctic regions. Results The Atlantic cod genome was shown to contain 14 globin genes, including nine hemoglobin genes organized in two unlinked clusters designated β5-α1-β1-α4 and β3-β4-α2-α3-β2. The diverged cod hemoglobin genes displayed different expression levels in adult fish, and tetrameric hemoglobins with or without a Root effect were predicted. The novel finding of maternally inherited hemoglobin mRNAs is consistent with a potential role played by fish hemoglobins in the non-specific immune response. In silico analysis of the six teleost genomes available showed that the two α-β globin clusters are flanked by paralogs of five duplicated genes, in agreement with the proposed teleost-specific duplication of the ancestral vertebrate globin cluster. Screening the genome of extant urochordate and cephalochordate species for conserved globin-flanking genes revealed linkage of RHBDF1, MPG and ARHGAP17 to globin genes in the tunicate Ciona intestinalis, while these genes together with LCMT are closely positioned in amphioxus (Branchiostoma floridae), but seem to be unlinked to the multiple globin genes identified in this species. Conclusion The plasticity of Atlantic cod to variable environmental conditions probably involves the expression of multiple globins with potentially different properties. The

  16. Phylogenetic Diversification of the Globin Gene Superfamily in Chordates

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    Summary Phylogenetic reconstructions provide a means of inferring the branching relationships among members of multigene families that have diversified via successive rounds of gene duplication and divergence. Such reconstructions can illuminate the pathways by which particular expression patterns and protein functions evolved. For example, phylogenetic analyses can reveal cases in which similar expression patterns or functional properties evolved independently in different lineages, either through convergence, parallelism, or evolutionary reversals. The purpose of this paper is to provide a robust phylogenetic framework for interpreting experimental data and for generating hypotheses about the functional evolution of globin proteins in chordate animals. To do this we present a consensus phylogeny of the chordate globin gene superfamily. We document the relative roles of gene duplication and whole-genome duplication in fueling the functional diversification of vertebrate globins, and we unravel patterns of shared ancestry among globin genes from representatives of the three chordate subphyla (Craniata, Urochordata, and Cephalochordata). Our results demonstrate the value of integrating phylogenetic analyses with genomic analyses of conserved synteny to infer the duplicative origins and evolutionary histories of globin genes. We also discuss a number of case studies that illustrate the importance of phylogenetic information when making inferences about the evolution of globin gene expression and protein function. Finally, we discuss why the globin gene superfamily presents special challenges for phylogenetic analysis, and we describe methodological approaches that can be used to meet those challenges. PMID:21557448

  17. Asynchronous DNA replication within the human. beta. -globin gene locus

    SciTech Connect

    Epner, E.; Forrester, W.C.; Groudine, M. )

    1988-11-01

    The timing of DNA replication of the human {beta}-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human {beta}-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-{gamma}-globin gene region and approximately 20 kilobases 5' to the {epsilon}-globin gene and 20 kilobases 3' to the {beta}-globin gene, replicate later and throughout S phase. A similar area is also present in the {alpha}-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks.

  18. Transcriptional promiscuity of the human /alpha/-globin gene

    SciTech Connect

    Whitelaw, E.; Hogben, P.; Hanscombe, O.; Proudfoot, N.J.

    1989-01-01

    The human /alpha/-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. The authors have investigated whether there is a relationship between these two observations. First, they investigated the mouse /alpha/-globin gene since it does not lie in an HTF island. They have demonstrated that it was not transcriptionally promiscuous. Second, they studied the transcriptional activity of the human /alpha/-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human /alpha/-globin gene. They found that in a nonreplicating system, the human //a/-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since they only observed enhancer independence of the human /alpha/-globin gene in a high-copy-number replicating system, they suggest that competition for trans-acting factors could explain these results. Finally, the authors' experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human /alpha/-globin cap site or within the gene itself.

  19. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. PMID:493112

  20. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed Central

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-01-01

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. Images PMID:493112

  1. Human fetal globin gene expression is regulated by LYAR

    PubMed Central

    Ju, Junyi; Wang, Ying; Liu, Ronghua; Zhang, Yichong; Xu, Zhen; Wang, Yadong; Wu, Yupeng; Liu, Ming; Cerruti, Loretta; Zou, Fengwei; Ma, Chi; Fang, Ming; Tan, Renxiang; Jane, Stephen M.; Zhao, Quan

    2014-01-01

    Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5′-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene. PMID:25092918

  2. Human fetal globin gene expression is regulated by LYAR.

    PubMed

    Ju, Junyi; Wang, Ying; Liu, Ronghua; Zhang, Yichong; Xu, Zhen; Wang, Yadong; Wu, Yupeng; Liu, Ming; Cerruti, Loretta; Zou, Fengwei; Ma, Chi; Fang, Ming; Tan, Renxiang; Jane, Stephen M; Zhao, Quan

    2014-09-01

    Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5'-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene. PMID:25092918

  3. Fetal Globin Gene Inducers: Novel Agents & New Potential

    PubMed Central

    Perrine, Susan P.; Castaneda, Serguei A.; Chui, David H.; Faller, Douglas V.; Berenson, Ronald J.; Fucharoen, Suthat

    2013-01-01

    Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide. PMID:20712788

  4. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

    NASA Astrophysics Data System (ADS)

    Lau, Yun-Fai; Kan, Yuet Wai

    1983-09-01

    We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

  5. [Joint locus of a/b-globin genes in Danio rerio is segregated into structural subdomains active at different stages of development].

    PubMed

    Dolgushin, K V; Petrova, N V; Iudinkova, E S; Razin, S V; Iarovaia, O V

    2015-01-01

    In the domain model of eukaryotic genome organization, the functional unit of the genome, along with the relevant regulatory elements, is considered to be a gene or a gene family. In hot-blooded vertebrate animals, the domains of a- and b-globin genes are positioned at different chromosomes and are organized and regulated in different fashion. In cold-blooded animals, in particular in tropical fish Danio rerio, a- and b globin genes are located in a common gene cluster. However, the joint a/b-globin gene cluster is subdivided into two development stage-specific subdomains, the adult one and the embryonic-larval one. In an attempt to find out whether this functional segregation correlates with structural segregation of the domain we compared the DNase I sensitivity and profiles of histone modifications of adult and embryonic-larval segments of the domain in cultured D. rerio fibroblasts. We have demonstrated that, in these nonerythroid cells, adult and embryonic- larval subdomains possess different DNase I sensitivities and different profiles of H3K27me3, a histone modification introduced by PRC2 complex. These observations suggest that joint a/b globin gene domain of Danio rerio is segregated into two structural subdomain harboring adult and embryonic-larval globin genes. PMID:26107904

  6. Duplication of the gamma-globin gene mediated by L1 long interspersed repetitive elements in an early ancestor of simian primates.

    PubMed Central

    Fitch, D H; Bailey, W J; Tagle, D A; Goodman, M; Sieu, L; Slightom, J L

    1991-01-01

    Regions surrounding the single gamma-globin gene of galago and the duplicated gamma 1- and gamma 2-globin genes of gibbon, rhesus monkey, and spider monkey were sequenced and aligned with those from humans. Contrary to previous studies, spider monkey was found to have not one but two gamma-globin genes, only one of which (gamma 2) is functional. The reconstructed evolutionary history of the gamma-globin genes and their flanking sequences traces their origin to a tandem duplication of a DNA segment approximately 5.5 kilobases long that occurred before catarrhine primates (humans, apes, and Old World monkeys) diverged from platyrrhines (New World monkeys), much earlier than previously thought. This reconstructed molecular history also reveals that the duplication resulted from an unequal homologous crossover between two related L1 long interspersed repetitive elements, one upstream and one downstream of the single ancestral gamma-globin gene. Perhaps facilitated by the redundancy resulting from the duplication, the gamma-globin genes escaped the selective constraints of embryonically functioning genes and evolved into fetally functioning genes. This view is supported by the finding that a burst of nonsynonymous substitutions occurred in the gamma-globin genes while they became restructured for fetal expression in the common ancestor of platyrrhines and catarrhines. PMID:1908094

  7. Butyrate Infusions in the Ovine Fetus Delay the Biologic Clock for Globin Gene Switching

    NASA Astrophysics Data System (ADS)

    Perrine, Susan P.; Rudolph, Abraham; Faller, Douglas V.; Roman, Christine; Cohen, Ruth A.; Chen, Shao-Jing; Kan, Yuet Wai

    1988-11-01

    The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal β -globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switching.

  8. Transcriptional activity of the human pseudogene psi alpha globin compared with alpha globin, its functional gene counterpart.

    PubMed Central

    Whitelaw, E; Proudfoot, N J

    1983-01-01

    Transcriptional analysis of the human pseudogene psi alpha globin has revealed the following features: (1) The promoter with a 23 bp deletion between the CCAAT and ATA boxes is functional both in vitro and in vivo, 3 fold and 10 fold less efficient, respectively, than alpha. (2) Both the psi alpha and alpha globin gene promoters are active in the absence of transcriptional enhancers, either a gene-encoded or viral enhancer. (3) The mutated poly(A) addition signal in psi alpha (AATGAA) appears to be completely nonfunctional. This result provides an explanation for the absence of psi alpha transcripts in human erythroid cells. Images PMID:6316269

  9. The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification

    PubMed Central

    2008-01-01

    Background Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. Results In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. Conclusion Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying

  10. Phylogenetic comparisons suggest that distance from the locus control region guides developmental expression of primate beta-type globin genes.

    PubMed

    Johnson, Robert M; Prychitko, Tom; Gumucio, Deborah; Wildman, Derek E; Uddin, Monica; Goodman, Morris

    2006-02-28

    Phylogenetic inferences drawn from comparative data on mammalian beta-globin gene clusters indicate that the ancestral primate cluster contained a locus control region (LCR) and five paralogously related beta-type globin loci (5'-LCR-epsilon-gamma-psieta-delta-beta-3'), with epsilon and gamma expressed solely during embryonic life. A gamma locus tandem duplication (5'-gamma(1)-gamma(2)-3') triggered gamma's evolution toward fetal expression but by a different trajectory in platyrrhines (New World monkeys) than in catarrhines (Old World monkeys and apes, including humans). In platyrrhine (e.g., Cebus) fetuses, gamma(1) at the ancestral distance from epsilon is down-regulated, whereas gamma(2) at increased distance is up-regulated. Catarrhine gamma(1) and gamma(2) acquired longer distances from epsilon (14 and 19 kb, respectively), and both are up-regulated throughout fetal life with gamma(1)'s expression predominating over gamma(2)'s. On enlarging the platyrrhine expression data, we find Aotus gamma is embryonic, Alouatta gamma is inactive at term, and in Callithrix, gamma(1) is down-regulated fetally, whereas gamma(2) is up-regulated. Of eight mammalian taxa now represented per taxon by embryonic, fetal, and postnatal beta-type globin gene expression data, four taxa are primates, and data for three of these primates are from this laboratory. Our results support a model in which a short distance (<10 kb) between epsilon and the adjacent gamma is a plesiomorphic character that allows the LCR to drive embryonic expression of both genes, whereas a longer distance (>10 kb) impedes embryonic activation of the downstream gene. PMID:16488971

  11. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    PubMed

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  12. Molecular nature of alpha-globin genes in the Saudi population

    PubMed Central

    Borgio, J. Francis

    2015-01-01

    Alpha-thalassemia (α-thal) is a disorder caused by the deletion of single or double α-globin genes, and/or point mutations in the α-globin genes. There are 2 common types of α-globin genes; HBA2 and HBA1. Recently, it has been discovered that the HBA2 gene is replaced by a unique HBA12 gene convert in 5.7% of the Saudi population. The α-globin genes have been emerging as a molecular target for the treatment of β-thalassemia (β-thal). Hence, it is essential to understand the molecular nature of α-globin genes to treat the most prevalent hemoglobin disorders, such as sickle cell disease, α-thal, and β-thal prevalent in the Kingdom of Saudi Arabia. Thirty-two different α-globin genotypes have been observed in the Saudi population. This review outlines the classification of the α-globin genes on the basis of their molecular nature and complex combinations of α-globin genes, and their variants predominant in Saudis. PMID:26593158

  13. Decitabine Increases Fetal Hemoglobin in P. Anubis by Increasing γ-globin Gene Transcription

    PubMed Central

    Akpan, Imo; Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective The mechanism responsible for increased fetal hemoglobin (HbF) levels following decitabine treatment remains controversial. These experiments were performed to evaluate the role of transcriptional versus translational mechanisms in the ability of decitabine to increase HbF levels in vivo. 2) Methods Three normal, nonanemic baboons were treated with decitabine subcutaneously (0.5mg/kg/d) for 10 days. The effect of decitabine on globin chain synthesis and globin mRNA levels was measured in pre- and post-treatment bone marrow (BM) aspirates by biosynthetic radiolabelling with [3H] leucine followed by separation of globin chains by HPLC, and real time PCR, respectively. The effect on DNA methylation of the ε- and γ-globin gene promoters was determined by bisulfite sequence analysis. 3) Results Decitabine treatment of normal, nonanemic baboons induced similar increases in the γ/γ+β chain synthetic ratio and the γ/total β-like globin RNA ratio and also increased expression of ε-globin transcripts. Increased expression of ε- and γ-globin was associated with decreased DNA methylation of the ε- and γ-globin gene promoters. 4) Conclusion Decitabine increases HbF in vivo by transcriptional activation of the γ-globin gene. PMID:20713129

  14. The 5' flanking region of human epsilon-globin gene.

    PubMed Central

    Baralle, F E; Shoulders, C C; Goodbourn, S; Jeffreys, A; Proudfoot, N J

    1980-01-01

    The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat. Images PMID:6253916

  15. Globin gene expression in correlation with G protein-related genes during erythroid differentiation

    PubMed Central

    2013-01-01

    Background The guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression. Results Human hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/μg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/μg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression). Conclusions These results

  16. Alpha globin gene analysis in a Sardinian family with interacting alpha and beta thalassaemia genes.

    PubMed

    Melis, M A; Galanello, R; Cao, A

    1983-04-01

    This paper reports the results of alpha globin gene analysis in a Sardinian family with interacting alpha and beta thalassaemia genes. The propositus, who was identified in a newborn survey as he had 26.0% Hb Bart's and 74.0% Hb F, successively developed the clinical and haematological picture of a transfusion-dependent thalassaemia major. According to the haemoglobin pattern, restriction endonuclease analysis of the DNA from this patient showed the deletion of three of the four alpha-globin structural genes. Thus beta 0-thalassaemia homozygotes with the delection of three alpha-structural genes seem to have a severe clinical phenotype similar to that of patients with a full complement of four alpha-globin structural genes. PMID:6299325

  17. Evolution of the globin gene family in deuterostomes: lineage-specific patterns of diversification and attrition.

    PubMed

    Hoffmann, Federico G; Opazo, Juan C; Hoogewijs, David; Hankeln, Thomas; Ebner, Bettina; Vinogradov, Serge N; Bailly, Xavier; Storz, Jay F

    2012-07-01

    In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available

  18. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

    PubMed

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  19. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

    PubMed Central

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  20. Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

    PubMed Central

    Cocca, E; Ratnayake-Lecamwasam, M; Parker, S K; Camardella, L; Ciaramella, M; di Prisco, G; Detrich, H W

    1995-01-01

    Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade. Images Fig. 2 Fig. 3 Fig. 4 PMID:7892183

  1. Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes.

    PubMed Central

    Grindlay, G J; Lanyon, W G; Allan, M; Paul, J

    1984-01-01

    Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin. Images PMID:6701091

  2. Transcription in vivo of an Alu family member upstream from the human epsilon-globin gene.

    PubMed Central

    Allan, M; Paul, J

    1984-01-01

    The more distal Alu repeat flanking the epsilon-globin gene is transcribed in K562 cells to generate transcripts 350-400 nucleotides in length. Initiation occurs at the start of the repeat, upstream of a putative PolIII control signal. These transcripts originate from the strand which does not code epsilon-globin and are oriented in the opposite direction from the gene. They are non-polyadenylated, nucleus-confined and are only detectable in association with expression of the epsilon-globin gene. Images PMID:6320117

  3. Identification and characterization of globin genes from two lepidopteran insects, Bombyx mori and Samia cynthia ricini.

    PubMed

    Kawaoka, Shinpei; Katsuma, Susumu; Meng, Yan; Hayashi, Nobumitsu; Mita, Kazuei; Shimada, Toru

    2009-02-15

    We describe the characterization of hemoglobin-like genes from two lepidopteran insects, Bombyx mori (Bmglobin) and Samia cynthia ricini (Scglobin). Bmglobin and Scglobin are predicted to be intracellular proteins and contain amino acids required for heme and oxygen binding. Expression profiles of two lepidopteran globins, especially Bmglobin, were different from that of other insect globins. Although other insect globins are mainly associated with the tracheal system, Bmglobin was expressed almost exclusively in the Malpighian tubules, and the strongest signal for Scglobin was detected in the fat body. Furthermore, biochemical fractionation analysis revealed that both Bmglobin and Scglobin were localized in the cytoplasm. These results suggest that each lepidopteran globin has a distinct role in the tissues in which it is expressed and that the functions of insect globins are more divergent than previously thought. PMID:19059317

  4. Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

    PubMed Central

    Villeponteau, B; Martinson, H

    1981-01-01

    A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts. Images PMID:6269092

  5. Understanding α-globin gene regulation and implications for the treatment of β-thalassemia.

    PubMed

    Mettananda, Sachith; Gibbons, Richard J; Higgs, Douglas R

    2016-03-01

    Over the past three decades, a vast amount of new information has been uncovered describing how the globin genes are regulated. This knowledge has provided significant insights into the general understanding of the regulation of human genes. It is now known that molecular defects within and around the α- and β-globin genes, as well as in the distant regulatory elements, can cause thalassemia. Unbalanced production of globin chains owing to defective synthesis of one, and the continued unopposed synthesis of another, is the central causative factor in the cellular pathology and pathophysiology of thalassemia. A large body of clinical, genetic, and experimental evidence suggests that altering globin chain imbalance by reducing the production of α-globin synthesis ameliorates the disease severity in patients with β-thalassemia. With the development of new genetic-based therapeutic tools that have a potential to decrease the expression of a selected gene in a tissue-specific manner, the possibility of decreasing expression of the α-globin gene to improve the clinical severity of β-thalassemia could become a reality. PMID:26695885

  6. Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome

    PubMed Central

    Opazo, Juan C.; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F.

    2015-01-01

    Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about

  7. Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

    PubMed Central

    2012-01-01

    Background The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation. Results We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified. The same approach was used to generate profile-2

  8. Correction of human. beta. sup S -globin gene by gene targeting

    SciTech Connect

    Shesely, E.G.; Hyungsuk Kim; Shehee, W.R.; Smithies, O. ); Papayannopoulou, T. ); Popovich, B.W. )

    1991-05-15

    As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

  9. Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells.

    PubMed

    Chou, Yu-Chi; Chen, Ruei-Lin; Lai, Zheng-Sheng; Song, Jen-Shin; Chao, Yu-Sheng; Shen, Che-Kun James

    2015-07-01

    Pharmacological induction of the fetal γ globin gene and the consequent formation of HbF (α2/γ2) in adult erythroid cells are one feasible therapeutic strategy for sickle cell disease (SCD) and severe β-thalassemias. Hydroxyurea (HU) is the current drug of choice for SCD, but serious side effects limit its clinical use. Moreover, 30 to 50% of patients are irresponsive to HU treatment. We have used high-throughput screening to identify benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and its derivatives (compounds I to VI) as potent γ globin inducers. Of the compounds, I to V exert superior γ globin induction and have better therapeutic potential than HU, likely because of their activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and modulation of expression levels and/or chromosome binding of γ globin gene regulators, including BCL11A, and chromatin structure over the γ globin promoter. Unlike sodium butyrate (NaB), the global levels of acetylated histones H3 and H4 are not changed by compound II treatment. Remarkably, compound II induces the γ globin gene in HU-resistant primary human adult erythroid cells, the p38 signaling pathway of which appears to be irresponsive to HU and NaB as well as compound II. This study provides a new framework for the development of new and superior compounds for treating SCD and severe β-thalassemias. PMID:25986606

  10. Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

    PubMed

    Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

    2014-05-01

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. PMID:24814285

  11. Repeated Evolution of Chimeric Fusion Genes in the β-Globin Gene Family of Laurasiatherian Mammals

    PubMed Central

    Gaudry, Michael J.; Storz, Jay F.; Butts, Gary Tyler; Campbell, Kevin L.; Hoffmann, Federico G.

    2014-01-01

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB “Lepore” deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived “anti-Lepore” duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20–100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. PMID:24814285

  12. Gene duplication, genome duplication, and the functional diversification of vertebrate globins

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    The functional diversification of the vertebrate globin gene superfamily provides an especially vivid illustration of the role of gene duplication and whole-genome duplication in promoting evolutionary innovation. For example, key globin proteins that evolved specialized functions in various aspects of oxidative metabolism and oxygen signaling pathways (hemoglobin [Hb], myoglobin [Mb], and cytoglobin [Cygb]) trace their origins to two whole-genome duplication events in the stem lineage of vertebrates. The retention of the proto-Hb and Mb genes in the ancestor of jawed vertebrates permitted a physiological division of labor between the oxygen-carrier function of Hb and the oxygen-storage function of Mb. In the Hb gene lineage, a subsequent tandem gene duplication gave rise to the proto α- and β-globin genes, which permitted the formation of multimeric Hbs composed of unlike subunits (α2β2). The evolution of this heteromeric quaternary structure was central to the emergence of Hb as a specialized oxygen-transport protein because it provided a mechanism for cooperative oxygen-binding and allosteric regulatory control. Subsequent rounds of duplication and divergence have produced diverse repertoires of α- and β-like globin genes that are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different stages of prenatal development and postnatal life. In the ancestor of jawless fishes, the proto Mb and Hb genes appear to have been secondarily lost, and the Cygb homolog evolved a specialized respiratory function in blood-oxygen transport. Phylogenetic and comparative genomic analyses of the vertebrate globin gene superfamily have revealed numerous instances in which paralogous globins have convergently evolved similar expression patterns and/or similar functional specializations in different organismal lineages. PMID:22846683

  13. A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

    PubMed

    Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

    2016-01-01

    Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase. PMID:27316998

  14. Distinctive Patterns of Evolution of the δ-Globin Gene (HBD) in Primates

    PubMed Central

    Moleirinho, Ana; Lopes, Alexandra M.; Seixas, Susana; Morales-Hojas, Ramiro; Prata, Maria J.; Amorim, António

    2015-01-01

    In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a

  15. Evolutionary pathway of pseudogenization of globin genes, α5 and β5, in genus Oryzias.

    PubMed

    Maruyama, Kouichi; Wang, Bing; Ishikawa, Yuji; Yasumasu, Shigeki; Iuchi, Ichiro

    2015-09-01

    Hemoglobin transports oxygen in many organisms and consists of α- and β-globin chains. Previously, using molecular phylogenetic analysis, we proposed that both α- and β-globins of teleost could be classified into four groups. We also showed that the Hd-rR strain of medaka (Oryzias latipes) inhabiting southern Japan had all four groups of globin genes but that the α- and β-globin genes of group III were pseudogenized (α5(ψα), β5(ψβ)). Based on the small degree of nucleotide variations, the pseudogenization of β5 was assumed to have occurred at a relatively late stage of evolution. Here, we compared the α5(ψα)-β5(ψβ) of two other strains of O. latipes and found that both α5(ψα) and β5(ψβ) of the northern Japanese and Korean strains were pseudogenized similar to those of Hd-rR. In a Philippine population (Oryzias luzonensis), α5(ψα) was also pseudogenized, but the structure was different from that of O. latipes, and β5(ψβ) was almost deleted. Interestingly, an Indonesian population (Oryzias celebensis) had α5 and β5 genes that were deduced to be functional. Indeed, they were expressed from the young to adult development stages, and this expression pattern was consistent with the expression of α2 and ad.α1 in Hd-rR. Because α2 and ad.α1 in Hd-rR were assigned to groups I and II, respectively, we speculate that their expression patterns might be altered by pseudogenization of group III genes. These results provide a basis for further investigations of recruiting and changing expression patterns of one globin gene after pseudogenization of other globin genes during evolution. PMID:26199047

  16. Evidence that the recently discovered theta 1-globin gene is functional in higher primates.

    PubMed

    Shaw, J P; Marks, J; Shen, C K

    A new subfamily of the alpha-globin-like family has recently been identified in higher primates, rabbit, galago and possibly the horse. One member of this subfamily, theta 1, is downstream from the adult alpha 1-globin gene. In orang-utan, but not in rabbit or galago, the theta 1-gene appears to be structurally intact, suggesting that it may be functional in this species. The orang-utan theta 1-gene possesses initiation and termination codons, and the predicted polypeptide differs from the orang-utan alpha 1-globin by 55 amino acids. The upstream promoter boxes CCAAT and ATA are present, although approximately 150 base pairs (bp) farther upstream than in the alpha 1-gene. This structural difference in the promoter between the orang-utan theta 1- and alpha 1-genes has led Proudfoot to speculate that the theta 1-gene may be inactive. We have now cloned the theta 1- and alpha 1-globin genes from the olive baboon, and have compared their sequences with those of orang-utan. The unique promoter structure of the orang-utan theta 1-gene is highly conserved in baboon, although the orang-utan and baboon diverged nearly 30 million years ago. The coding sequences of the two theta 1-genes differ by only 6.3% with 22 out of 27 nucleotide substitutions being codon third position silent changes. These data support the view that the theta 1-gene has been functional in the baboon, orang-utan, and by implication, in man. We also estimate that the duplication event generating the theta 1- and alpha-globin-like subfamilies may have occurred as much as 260 million years ago. PMID:3561513

  17. Evidence on primate phylogeny from epsilon-globin gene sequences and flanking regions.

    PubMed

    Porter, C A; Sampaio, I; Schneider, H; Schneider, M P; Czelusniak, J; Goodman, M

    1995-01-01

    Phylogenetic relationships among various primate groups were examined based on sequences of epsilon-globin genes. epsilon-globin genes were sequenced from five species of strepsirhine primates. These sequences were aligned and compared with other known primate epsilon-globin sequences, including data from two additional strepsirhine species, one species of tarsier, 19 species of New World monkeys (representing all extant genera), and five species of catarrhines. In addition, a 2-kb segment upstream of the epsilon-globin gene was sequenced in two of the five strepsirhines examined. This upstream sequence was aligned with five other species of primates for which data are available in this segment. Domestic rabbit and goat were used as outgroups. This analysis supports the monophyly of order Primates but does not support the traditional prosimian grouping of tarsiers, lorisoids, and lemuroids; rather it supports the sister grouping of tarsiers and anthropoids into Haplorhini and the sister grouping of lorisoids and lemuroids into Strepsirhini. The mouse lemur (Microcebus murinus) and dwarf lemur (Cheirogaleus medius) appear to be most closely related to each other, forming a clade with the lemuroids, and are probably not closely related to the lorisoids, as suggested by some morphological studies. Analysis of the epsilon-globin data supports the hypothesis that the aye-aye (Daubentonia madagascariensis) shares a sister-group relationship with other Malagasy strepsirhines (all being classified as lemuroids). Relationships among ceboids agree with findings from a previous epsilon-globin study in which fewer outgroup taxa were employed. Rates of molecular evolution were higher in lorisoids than in lemuroids. PMID:7714911

  18. β-Thalassemia Due to Intronic LINE-1 Insertion in the β-Globin Gene (HBB): Molecular Mechanisms Underlying Reduced Transcript Levels of the β-GlobinL1 Allele

    PubMed Central

    Lanikova, Lucie; Kucerova, Jana; Indrak, Karel; Divoka, Martina; Issa, Jean-Pierre; Papayannopoulou, Thalia; Prchal, Josef T.; Divoky, Vladimir

    2016-01-01

    We describe the molecular etiology of β+-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globinL1 allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globinL1 allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globinL1 transcription despite permanent β-globinL1 promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globinL1 allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia. PMID:23878091

  19. A Novel Mutation in the Promoter Region of the β-Globin Gene: HBB: c.-127G > C.

    PubMed

    Bilgen, Turker; Canatan, Duran; Delibas, Serpil; Keser, Ibrahim

    2016-08-01

    Novel β-globin gene mutations are still occasionally being reported, especially when evaluating milder phenotypes. We report here a novel putative mutation in the promoter region of the β-globin gene and assess its clinical implications. A family, parents and four siblings, with hematological and clinical features suspected of being β-globin gene mutation(s), were involved in this study. In addition to hematological and clinical evaluations of the whole family, molecular analyses of the β-globin gene were performed by direct sequencing. Sequencing of the β-globin gene revealed a novel genomic alteration in the regulatory region of the gene. This novel genomic alteration was defined as HBB: c.-127G > C according to the Human Genome Variation Society (HGVS) nomenclature. Two siblings were found to be carriers of the HBB: c.-127G > C mutation, while the other two siblings were carriers of the codon 8 (-AA) (HBB: c.25_26delAA) deletion of the β-globin gene. The mother was a compound heterozygote for the codon 8 and HBB: c.-127G > C mutations. Based on hematological and clinical evaluations, we conclude that this novel β-globin gene promoter region change would be associated with a mild phenotype of β-thalassemia (β-thal). PMID:27349616

  20. A phylogenomic profile of globins

    PubMed Central

    Vinogradov, Serge N; Hoogewijs, David; Bailly, Xavier; Arredondo-Peter, Raúl; Gough, Julian; Dewilde, Sylvia; Moens, Luc; Vanfleteren, Jacques R

    2006-01-01

    Background Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold. Results A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. Conclusion Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals. PMID:16600051

  1. Beta-globin gene cluster haplotype frequencies in Khalkhs and Buryats of Mongolia.

    PubMed

    Shimizu, Koji; Tokimasa, Kozue; Takeuchi, Yukiko; Gereksaikhan, Tudevdagva; Tanabe, Yuichi; Omoto, Keiichi; Imanishi, Tadashi; Harihara, Shinji; Hao, Luping; Jing, Feng

    2006-12-01

    Beta-globin gene cluster haplotype frequencies of 169 Khalkhs and 145 Buryats were estimated, and their characteristics were compared with those of Evenkis, Oroqens, Koreans, Japanese, and three Colombian Amerindian groups. The present study suggests that Colombian Amerindians diverged first from Asian populations and then Buryats diverged from other Asian populations. PMID:17564253

  2. Molecular cloning and expression of α-globin and β-globin genes from crocodile (Crocodylus siamensis).

    PubMed

    Anwised, Preeyanan; Kabbua, Thai; Temsiripong, Theeranan; Dhiravisit, Apisak; Jitrapakdee, Sarawut; Araki, Tomohiro; Yoneda, Kazunari; Thammasirirak, Sompong

    2013-03-01

    The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein. PMID:23463382

  3. Nonsense mutations in the human. beta. -globin gene affect mRNA metabolism

    SciTech Connect

    Baserga, S.J.; Benz, E.J. Jr. )

    1988-04-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.

  4. Recombination within and between the human insulin and beta-globin gene loci.

    PubMed Central

    Lebo, R V; Chakravarti, A; Buetow, K H; Cheung, M C; Cann, H; Cordell, B; Goodman, H

    1983-01-01

    We detected a large number of polymorphic insulin restriction fragments in black Americans. These different size fragments were probably generated by unequal recombination on both sides of the human insulin gene. Population genetic analysis indicates that recombination occurred 33 times more frequently than expected to generate this large number of polymorphic fragments. Specific properties of the unique repeated 14- to 16-base-pair sequences 5' to the insulin gene suggest that this sequence would promote increased unequal recombination. Additional pedigree analysis showed that the recombination rate between the structural insulin and beta-globin gene loci was 14% with strong evidence for linkage. Since both insulin and beta-globin have been mapped to the short arm of human chromosome 11, this study establishes that the genetic map distance between these genes is 14.2 centimorgans. PMID:6348773

  5. Oral decitabine reactivates expression of the methylated gamma-globin gene in Papio anubis.

    PubMed

    Lavelle, Donald; Chin, Janet; Vaitkus, Kestis; Redkar, Sanjeev; Phiasivongsa, Pasit; Tang, Chunlin; Will, Roselle; Hankewych, Maria; Roxas, Bryan; Singh, Mahipal; Saunthararajah, Yogen; Desimone, Joseph

    2007-11-01

    The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-2'-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated gamma-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated gamma-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the epsilon- and gamma-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis). PMID:17696208

  6. Characterization of two globin genes from the malaria mosquito Anopheles gambiae: divergent origin of nematoceran haemoglobins.

    PubMed

    Burmester, Thorsten; Klawitter, Sabine; Hankeln, Thomas

    2007-04-01

    The chironomid midges are the only insects that harbour true haemoglobin in their haemolymph. Here we report the identification of haemoglobin genes in two other nematoceran species. Two paralogous haemoglobin genes (glob1 and glob2) from the malaria mosquito Anopheles gambiae were cloned and sequenced. Furthermore, we identified two orthologous haemoglobin genes in the yellow fever mosquito Aedes aegypti. All four haemoglobins were predicted to be intracellular proteins, with the amino acids required for heme- and oxygen-binding being conserved. In situ-hybridization studies showed that glob1 and glob2 expression in An. gambiae is mainly associated with the tracheal system. This pattern resembles that of other insect intracellular globins. We also observed expression of glob2 in visceral muscles. Phylogenetic analyses showed that the globins of the mosquitoes and the Chironomidae are not orthologous. The chironomid haemoglobins share a recent common origin with the brachyceran glob1 proteins. The mosquito glob1 and glob2 proteins, which separated by gene duplication around 170 million years ago, form a distinct clade of more ancient evolutionary origin within the insects. The glob1 genes have introns in the ancestral globin positions B12.2 and G7.0. An additional intron was observed in Ae. aegypti glob1 helix position E18.0, providing evidence for a recent intron gain event. Both mosquito glob2 genes have lost the B12.2 intron. This pattern must be interpreted in terms of dynamic intron gain and loss events in the globin gene lineage. PMID:17298561

  7. Association of Xmn I Polymorphism and Hemoglobin E Haplotypes on Postnatal Gamma Globin Gene Expression in Homozygous Hemoglobin E

    PubMed Central

    Ekwattanakit, Supachai; Monteerarat, Yuwarat; Riolueang, Suchada; Tachavanich, Kalaya; Viprakasit, Vip

    2012-01-01

    Background and Objectives. To explore the role of cis-regulatory sequences within the β globin gene cluster at chromosome 11 on human γ globin gene expression related to Hb E allele, we analyze baseline hematological data and Hb F values together with β globin haplotypes in homozygous Hb E. Patients and Methods. 80 individuals with molecularly confirmed homozygous Hb E were analyzed for the β globin haplotypes and Xmn I polymorphism using PCR-RFLPs. 74 individuals with complete laboratory data were further studied for association analyses. Results. Eight different β globin haplotypes were found linked to Hb E alleles; three major haplotypes were (a) (III), (b) (V), and (c) (IV) accounting for 94% of Hb E chromosomes. A new haplotype (Th-1) was identified and most likely converted from the major ones. The majority of individuals had Hb F < 5%; only 10.8% of homozygous Hb E had high Hb F (average 10.5%, range 5.8–14.3%). No association was found on a specific haplotype or Xmn I in these individuals with high Hb F, measured by alkaline denaturation. Conclusion. The cis-regulation of γ globin gene expression might not be apparent under a milder condition with lesser globin imbalance such as homozygous Hb E. PMID:23049556

  8. Precise nucleosome positioning in the promoter of the chicken beta A globin gene.

    PubMed

    Kefalas, P; Gray, F C; Allan, J

    1988-01-25

    Histone octamers were reconstituted onto 5' end-labelled DNA fragments derived from the promoter region of the chicken beta A globin gene. The location of the reconstituted histone octamer with respect to the DNA sequence of each fragment was assessed by Exonuclease III digestion of purified nucleosome monomers. By this approach we have found a strong preference for histone octamers to be positioned over nucleotides -206 to -62 relative to the gene cap site. This stretch of DNA contains all those 5' beta globin sequences which, by DNase footprinting, bind specific protein factors and incorporates three promoter consensus sequence motifs. The upstream terminal 32 base pairs of this DNA segment contains the binding sites for the erythrocyte specific G-string binding protein and transcription factor Spl and appears to be relatively weakly bound to the histone octamer. PMID:3340546

  9. Precise nucleosome positioning in the promoter of the chicken beta A globin gene.

    PubMed Central

    Kefalas, P; Gray, F C; Allan, J

    1988-01-01

    Histone octamers were reconstituted onto 5' end-labelled DNA fragments derived from the promoter region of the chicken beta A globin gene. The location of the reconstituted histone octamer with respect to the DNA sequence of each fragment was assessed by Exonuclease III digestion of purified nucleosome monomers. By this approach we have found a strong preference for histone octamers to be positioned over nucleotides -206 to -62 relative to the gene cap site. This stretch of DNA contains all those 5' beta globin sequences which, by DNase footprinting, bind specific protein factors and incorporates three promoter consensus sequence motifs. The upstream terminal 32 base pairs of this DNA segment contains the binding sites for the erythrocyte specific G-string binding protein and transcription factor Spl and appears to be relatively weakly bound to the histone octamer. Images PMID:3340546

  10. Characteristic beta-globin gene cluster haplotypes of Evenkis and Oroqens in north China.

    PubMed

    Shimizu, Koji; Marubayashi, Azusa; Tokimasa, Kozue; Harihara, Shinji; Omoto, Keiichi; Imanishi, Tadashi; Hao, Luping; Jin, Feng

    2004-10-01

    Haplotype frequencies of the beta-globin gene cluster were estimated for 114 Evenkis and 81 Oroqens from northeast China, and their characteristics were compared with those in Japanese, Koreans, and three Colombian Amerindian groups of South America (Wayuu, Kamsa, and Inga tribes). A major 5' subhaplotype (5' to the delta-globin gene) was + - - - - in Evenkis, whereas + - - - -, - + + - +, and - + - + + were the major subhaplotypes in Oroqens. One possible candidate for an ancestral 5' subhaplotype, - - - - -, was found in one Evenki (0.5%) and three Oroqen chromosomes (2.0%). They were observed as heterozygous forms for + ---- and -----. Major haplotypes were +-----+, + -----+-, and + - - - - + + in Evenkis, whereas they were +-----+,-++-+-+, +----+-, and -+-++-+ in Oroqens. The lowest Nei's genetic distance values of Evenkis or Oroqens based on the 5' subhaplotype frequency distributions were observed in relation to the Wayuu or Koreans, respectively, but those of Evenkis and Oroqens based on the haplotype frequency distributions were found in relation to Koreans. PMID:15757246

  11. Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site▿ †

    PubMed Central

    Harju-Baker, Susanna; Costa, Flávia C.; Fedosyuk, Halyna; Neades, Renee; Peterson, Kenneth R.

    2008-01-01

    Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ɛ-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the −566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the −566/−567 GATA sites of the proximal γ-globin promoters. PMID:18347053

  12. Analysis of α1 and α2 globin genes among patients with hemoglobin Adana in Malaysia.

    PubMed

    Lee, T Y; Lai, M I; Ismail, P; Ramachandran, V; Tan, J A M A; Teh, L K; Othman, R; Hussein, N H; George, E

    2016-01-01

    Hemoglobin (Hb) Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a non-deletional α-thalassemia variant found in Malaysia. An improvement in the molecular techniques in recent years has made identification of Hb Adana much easier. For this study, a total of 26 Hb Adana α-thalassemia intermedia and 10 Hb Adana trait blood samples were collected from patients. Common deletional and non-deletional α-thalassemia genotypes were determined using multiplex gap polymerase chain reaction (PCR) and multiplex ARMS PCR techniques. Identification of the Hb Adana location on the α-globin gene was carried out using genomic sequencing and the location of the mutation was confirmed via restriction fragment length polymorphism-PCR. Among the 36 samples, 24 (66.7%) had the -α(3.7)/α(Cd59)α mutation, while the -α(3.7)/α(Cd59)α mutation accounted for 2 samples (5.6%) and the remaining 10 (27.8%) samples were α/α(Cd59)α. All 36 samples were found to have the Hb Adana mutation on the α2-globin gene. The position of the α-globin gene mutation found in our cases was similar to that reported in Indonesia (16%) but not to that in Turkey (0.6%). Our results showed that the Hb Adana mutation was preferentially present in the α2-globin genes in Malays compared to the other ethnicities in Malaysia. Thus, the Malays might have similar ancestry based on the similarities in the Hb Adana position. PMID:27173219

  13. Thalassaemia mutations within the 5'UTR of the human beta-globin gene disrupt transcription.

    PubMed

    Sgourou, Argyro; Routledge, Samantha; Antoniou, Michael; Papachatzopoulou, Adamantia; Psiouri, Lambrini; Athanassiadou, Aglaia

    2004-03-01

    The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment. PMID:15009072

  14. Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

    PubMed Central

    Storz, Jay F.; Natarajan, Chandrasekhar; Cheviron, Zachary A.; Hoffmann, Federico G.; Kelly, John K.

    2012-01-01

    Spatially varying selection on a given polymorphism is expected to produce a localized peak in the between-population component of nucleotide diversity, and theory suggests that the chromosomal extent of elevated differentiation may be enhanced in cases where tandemly linked genes contribute to fitness variation. An intriguing example is provided by the tandemly duplicated β-globin genes of deer mice (Peromyscus maniculatus), which contribute to adaptive differentiation in blood–oxygen affinity between high- and low-altitude populations. Remarkably, the two β-globin genes segregate the same pair of functionally distinct alleles due to a history of interparalog gene conversion and alleles of the same functional type are in perfect coupling-phase linkage disequilibrium (LD). Here we report a multilocus analysis of nucleotide polymorphism and LD in highland and lowland mice with different genetic backgrounds at the β-globin genes. The analysis of haplotype structure revealed a paradoxical pattern whereby perfect LD between the two β-globin paralogs (which are separated by 16.2 kb) is maintained in spite of the fact that LD within both paralogs decays to background levels over physical distances of less than 1 kb. The survey of nucleotide polymorphism revealed that elevated levels of altitudinal differentiation at each of the β-globin genes drop away quite rapidly in the external flanking regions (upstream of the 5′ paralog and downstream of the 3′ paralog), but the level of differentiation remains unexpectedly high across the intergenic region. Observed patterns of diversity and haplotype structure are difficult to reconcile with expectations of a two-locus selection model with multiplicative fitness. PMID:22042573

  15. Coinheritance of a Rare Nucleotide Substitution on the β-Globin Gene and Other Known Mutations in the Globin Clusters: Management in Genetic Counseling.

    PubMed

    Vinciguerra, Margherita; Passarello, Cristina; Leto, Filippo; Crivello, Anna; Fustaneo, Maria; Cassarà, Filippo; Cannata, Monica; Maggio, Aurelio; Giambona, Antonino

    2016-08-01

    A large number of methods for DNA analysis are available to identify defects in globin genes associated with hemoglobin (Hb) disorders. In this study, we report a rare nucleotide (nt) substitution on the β-globin gene, nt 781 in the second intron [IVS-II-781 (C > G); HBB: c.316-70C > G], identified in four patients. This nt substitution was previously described only as a personal communication to the HbVar database and indicated as a β(0) or β(+) mutation. The purpose of this study was to evaluate the clinical implication of this nt change, particularly when coinherited with severe β-thalassemia (β-thal), in order to be able to conduct appropriate genetic counseling. Genetic studies were performed on two subjects, one carried Hb S [β6(A3)Glu→Val; HBB: c.20A > T], and the other carried IVS-I-110 (G > A) (HBB: c.93-21G > A). All these subjects showed this new β nt substitution in association with Hb A2' (or Hb B2) [δ16(A13)Gly→Arg; HBD: c.49G > C]. Another 16 samples, carrying the same δ variant as the probands, were processed by β-globin gene sequencing in order to better understand the correlation between this Hb variant and the rare nt substitution reported in this study. The present investigation emphasizes the importance of sharing the observed nt changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression, the possible interactions with known molecular defects and to formulate appropriate genetic counseling for at-risk couples. PMID:27258795

  16. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the β(A(T87Q))-Globin Gene.

    PubMed

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-02-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (β(AT87Q)-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. β(AT87Q)-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

  17. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the βA(T87Q)-Globin Gene

    PubMed Central

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-01-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (βAT87Q-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. βAT87Q-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

  18. Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

    PubMed Central

    Shani, M

    1986-01-01

    A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice. Images PMID:3023942

  19. Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

    PubMed Central

    Kemper, B; Jackson, P D; Felsenfeld, G

    1987-01-01

    We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared. Images PMID:3600658

  20. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

    PubMed Central

    Sadelain, M; Wang, C H; Antoniou, M; Grosveld, F; Mulligan, R C

    1995-01-01

    Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of

  1. S1 nuclease analysis of alpha-globin gene expression in preleukemic patients with acquired hemoglobin H disease after transfer to mouse erythroleukemia cells.

    PubMed

    Helder, J; Deisseroth, A

    1987-04-01

    The loss of alpha-globin gene transcriptional activity rarely occurs as an acquired abnormality during the evolution of myeloproliferative disease or preleukemia. To test whether the mutation responsible for the loss of alpha-globin gene expression (hemoglobin H disease) in these patients is linked with the alpha-globin genes on chromosome 16, we transferred chromosome 16 from preleukemic patients with acquired hemoglobin H disease to mouse erythroleukemia cells and measured the transcriptional activity of the human alpha-globin genes. After transfer to mouse erythroleukemia cells, the expression of human alpha-globin genes from the peripheral blood or marrow cells of preleukemic patients with acquired hemoglobin H disease was similar to that of human alpha-globin genes transferred to mouse erythroleukemia cells from normal donors. These data showed that factor(s) in the mouse erythroleukemia cell can genetically complement the alpha-globin gene defect in these preleukemia patients with acquired hemoglobin H disease and suggest that altered expression of a gene in trans to the alpha-globin gene may be responsible for the acquisition of hemoglobin H disease in these patients. PMID:3031681

  2. Origin and Ascendancy of a Chimeric Fusion Gene: The β/δ-Globin Gene of Paenungulate Mammals

    PubMed Central

    Opazo, Juan C.; Sloan, Angela M.; Campbell, Kevin L.

    2009-01-01

    The δ-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked β-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric β/δ fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the β/δ fusion gene in elephants is structurally similar to the “anti-Lepore” duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric β/δ fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the β-chain subunits of adult hemoglobin. A phylogenetic survey of β-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric β/δ fusion gene and the concomitant inactivation of the HBB gene predated the radiation of “Paenungulata,” a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived β/δ fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal δ/β fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin. PMID:19332641

  3. Same. beta. -globin gene mutation is present on nine different. beta. -thalassemia chromosomes in a Sardinian population

    SciTech Connect

    Pirastu, M.; Galanello, R.; Doherty, M.A.; Tuveri, T.; Cao, A.; Kan, Y.W.

    1987-05-01

    The predominant ..beta..-thalassemia in Sardinia is the ..beta../sup 0/ type in which no ..beta..-globin chains are synthesized in the homozygous state. The authors determined the ..beta..-thalassemia mutations in this population by the oligonucleotide-probe method and defined the chromosome haplotypes on which the mutation resides. The same ..beta../sup 39(CAG..-->..TAG)/ nonsense mutation was found on nine different chromosome haplotypes. Although this mutation may have arisen more than once, the multiple haplotypes could also be generated by crossing over and gene conversion events. These findings underscore the frequency of mutational events in the ..beta..-globin gene region.

  4. Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

    PubMed Central

    Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

    1995-01-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. Images Figure 2 Figure 3 PMID:8533755

  5. Human {beta}-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old

    SciTech Connect

    Beraud-Colomb, E. |; Maroc, N.; Roubin, R.

    1995-12-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the P-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for P-globin frameworks by sequencing through two variable positions and for a polymorphic (AT){sub x}(T){sub y} microsatellite 500 bp upstream of the P-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. 34 refs., 3 figs., 2 tabs.

  6. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

    SciTech Connect

    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-06-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes.

  7. Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

    PubMed

    Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

    2009-05-27

    Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia. PMID:19371028

  8. Genomic organization and differential signature of positive selection in the alpha and beta globin gene clusters in two cetacean species.

    PubMed

    Nery, Mariana F; Arroyo, José Ignacio; Opazo, Juan C

    2013-01-01

    The hemoglobin of jawed vertebrates is a heterotetramer protein that contains two α- and two β-chains, which are encoded by members of α- and β-globin gene families. Given the hemoglobin role in mediating an adaptive response to chronic hypoxia, it is likely that this molecule may have experienced a selective pressure during the evolution of cetaceans, which have to deal with hypoxia tolerance during prolonged diving. This selective pressure could have generated a complex history of gene turnover in these clusters and/or changes in protein structure themselves. Accordingly, we aimed to characterize the genomic organization of α- and β-globin gene clusters in two cetacean species and to detect a possible role of positive selection on them using a phylogenetic framework. Maximum likelihood and Bayesian phylogeny reconstructions revealed that both cetacean species had retained a similar complement of putatively functional genes. For the α-globin gene cluster, the killer whale presents a complement of genes composed of HBZ, HBK, and two functional copies of HBA and HBQ genes, whereas the dolphin possesses HBZ, HBK, HBA and HBQ genes, and one HBA pseudogene. For the β-globin gene cluster, both species retained a complement of four genes, two early expressed genes-HBE and HBH-and two adult expressed genes-HBD and HBB. Our natural selection analysis detected two positively selected sites in the HBB gene (56 and 62) and four in HBA (15, 21, 49, 120). Interestingly, only the genes that are expressed during the adulthood showed the signature of positive selection. PMID:24259315

  9. Total beta-globin gene deletion has high frequency in Filipinos

    SciTech Connect

    Patrick, N.; Miyakawa, F.; Hunt, J.A.

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  10. Evaluation of α-Globin Gene Mutations Among Different Ethnic Groups in Khuzestan Province, Southwest Iran.

    PubMed

    Khosravi, Abbas; Jalali-Far, Mohammadali; Saki, Najmaldin; Hosseini, Hossein; Galehdari, Hamid; Kiani-Ghalesardi, Omid; Paridar, Mostafa; Azarkeivan, Azita; Magaji-Hamid, Kabir

    2016-01-01

    α-Thalassemia (α-thal) is one of the most common inherited hemoglobin (Hb) disorders in the world. In addition to large deletions, over 50 different α-thal point mutations were detected around the world, thus, patients showed different phenotypes with regard to genotype. This study evaluated the genetic frequency of α-thal in Khuzestan Province, Southwest Iran, to help implement premarital and prenatal screening programs. The study was conducted on couples proposing to get married and parents who were referred to the genetic center of Shafa Hospital, Ahvaz, Iran, for prenatal diagnosis (PND) in 2012. Genomic DNA was purified by the salting-out method and tested using multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system-PCR (ARMS-PCR), reverse hybridization test strips and DNA sequencing. Overall, 11 mutations were found on the α-globin genes. Based on gene frequency, the most common mutant allele was -α(3.7) (rightward) (71.3%) followed by the two gene deletion - -(MED) (9.7%). Other common mutations were α(codon 19)α (GCG>GC-, α2) (8.4%), the polyadenylation (polyA1) site α(polyA1)α (AATAAA>AATAAG) (2.8%), and α(-5 nt)α (-TGAGG) (2.0%). In addition, an extremely rare mutation at α(codon 21)α [Hb Fontainebleau, HBA2: c.64G > C (or HBA1)] was also found. The results of this study are critical for correct diagnosis of α-thal carriers, premarriage counseling and PND. This study suggests that the distribution of mutations on the α-globin genes differs among the ethnic groups in Khuzestan Province as well as in other provinces. PMID:26878087

  11. Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

    PubMed Central

    Gong, Q H; McDowell, J C; Dean, A

    1996-01-01

    Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free

  12. Characterization of a large deletion in the {beta}-globin gene cluster in a newborn with hemoglobin FE

    SciTech Connect

    Louie, E.; Dietz, L.; Shafer, F.

    1994-09-01

    A sample on a newborn with hemoglobin FE screen results was obtained to investigate whether E/E or B/{beta}{degrees} thalassemia was present using polymerase chain reaction (PCR) methodology. The newborn appeared homozygous for the hemoglobin E mutation in our initial study, but the parents` genotypes did not support this diagnosis. The father is homozygous for the absence of the hemoglobin E mutation (non E/non E) and the mother is heterozygous (E/non E) for this mutation. The limitation of PCR analysis is an assumption that the amplification of the two {beta}-globin alleles is equivalent. A large deletion on one {beta}-globin gene, which would produce E/{beta}{degrees} thalassemia, would be missed if it included part or the entire region subjected to amplification. The family results were consistent with either non-paternity, sample mix-up or such a deletion of the {beta}-globin gene in the father and child. To rule out the possibility of non-paternity, two polymorphic loci (HLA on chromosome 6 and a VNTR system of chromosome 17) that are outside of the {beta}-globin gene were analyzed and show that inheritance is consistent and the likelihood of a sample mix-up is then reduced. We therefore believe there is a gene deletion in this family. At the present time, analyses of the RFLPs that are 5{prime} of the {beta}-globin gene cluster show that the polymorphisms most distal from the 5{prime} {beta}-globin gene are not being inherited as expected. These results support our interpretation that a deletion exists in the father and was inherited by the child. The father`s clinical picture of possible HPFH (the father has 12% hemoglobin F) also supports the interpretation of a deletion in this family. Deletions of the {beta}-globin gene within this ethnic group are rare. Currently, Southern blots on the family are being probed to determine the extent of the putative deletion.

  13. Genomic Organization and Differential Signature of Positive Selection in the Alpha and Beta Globin Gene Clusters in Two Cetacean Species

    PubMed Central

    Nery, Mariana F.; Arroyo, José Ignacio; Opazo, Juan C.

    2013-01-01

    The hemoglobin of jawed vertebrates is a heterotetramer protein that contains two α- and two β-chains, which are encoded by members of α- and β-globin gene families. Given the hemoglobin role in mediating an adaptive response to chronic hypoxia, it is likely that this molecule may have experienced a selective pressure during the evolution of cetaceans, which have to deal with hypoxia tolerance during prolonged diving. This selective pressure could have generated a complex history of gene turnover in these clusters and/or changes in protein structure themselves. Accordingly, we aimed to characterize the genomic organization of α- and β-globin gene clusters in two cetacean species and to detect a possible role of positive selection on them using a phylogenetic framework. Maximum likelihood and Bayesian phylogeny reconstructions revealed that both cetacean species had retained a similar complement of putatively functional genes. For the α-globin gene cluster, the killer whale presents a complement of genes composed of HBZ, HBK, and two functional copies of HBA and HBQ genes, whereas the dolphin possesses HBZ, HBK, HBA and HBQ genes, and one HBA pseudogene. For the β-globin gene cluster, both species retained a complement of four genes, two early expressed genes—HBE and HBH—and two adult expressed genes—HBD and HBB. Our natural selection analysis detected two positively selected sites in the HBB gene (56 and 62) and four in HBA (15, 21, 49, 120). Interestingly, only the genes that are expressed during the adulthood showed the signature of positive selection. PMID:24259315

  14. The relationship between the presence of extra α-globin genes and blood cell traits in Altamurana sheep

    PubMed Central

    2003-01-01

    Additional α-globin genes in sheep might produce extra α-globin chains and, consequently, the subject carrying triplicated (ααα) or quadruplicated (αααα) haplotypes may exhibit different hematological phenotypes when compared to the normal duplicated (αα) homozygotes (NN). Both ααα and αααα heterozygous (ND) and ααα and αααα homozygous (DD) individuals were obtained by selection and inbreeding. Chromatographic RP-HPLC analyses of the globin chains of 65 subjects (15 DD, 20 ND and 30 NN) were performed. A highly significant linear regression (r2 = 0.967) of the α/β ratio on the number of α-globin genes was found, and the α/β ratio ranged on average from 1.0 in NN individuals to 1.2 in the ND and 1.6 in the DD subjects. Values for blood fell within the range of normality but were rather peculiar as a whole. When the erythrocytes of individuals carrying normal arrangements were compared with those of subjects with extra α-genes, the latter had fewer erythrocytes that were bigger in size and had a higher Hb content and a greater osmotic fragility. This hematological picture is consistent with the existence of an unbalanced α/β ratio. PMID:12927085

  15. HbVar: A relational database of human hemoglobin variants and thalassemia mutations at the globin gene server.

    PubMed

    Hardison, Ross C; Chui, David H K; Giardine, Belinda; Riemer, Cathy; Patrinos, George P; Anagnou, Nicholas; Miller, Webb; Wajcman, Henri

    2002-03-01

    We have constructed a relational database of hemoglobin variants and thalassemia mutations, called HbVar, which can be accessed on the web at http://globin.cse.psu.edu. Extensive information is recorded for each variant and mutation, including a description of the variant and associated pathology, hematology, electrophoretic mobility, methods of isolation, stability information, ethnic occurrence, structure studies, functional studies, and references. The initial information was derived from books by Dr. Titus Huisman and colleagues [Huisman et al., 1996, 1997, 1998]. The current database is updated regularly with the addition of new data and corrections to previous data. Queries can be formulated based on fields in the database. Tables of common categories of variants, such as all those involving the alpha1-globin gene (HBA1) or all those that result in high oxygen affinity, are maintained by automated queries on the database. Users can formulate more precise queries, such as identifying "all beta-globin variants associated with instability and found in Scottish populations." This new database should be useful for clinical diagnosis as well as in fundamental studies of hemoglobin biochemistry, globin gene regulation, and human sequence variation at these loci. PMID:11857738

  16. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    PubMed Central

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin muα-globin2/huβ-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

  17. Gene expression analysis of the embryonic subplate

    PubMed Central

    Oeschger, Franziska M.; Wang, Wei-Zhi; Lee, Sheena; García-Moreno, Fernando; Goffinet, André M.; Arbones, Mariona; Rakic, Sonia; Molnár, Zoltán

    2015-01-01

    The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later stages, they are involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared to the cortical plate at this stage. Using quantitative RT-PCR, in situ hybridization and immunohistochemistry, we have confirmed specific expression in the E15.5 subplate for 13 selected genes which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 out of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to the maturation and electrophysiological properties of subplate cells and to axonal growth and guidance. PMID:21862448

  18. Gene expression analysis of the embryonic subplate.

    PubMed

    Oeschger, Franziska M; Wang, Wei-Zhi; Lee, Sheena; García-Moreno, Fernando; Goffinet, André M; Arbonés, Maria L; Rakic, Sonja; Molnár, Zoltán

    2012-06-01

    The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later developmental stages, they are predominantly involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day (E) 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared with the cortical plate at this stage. Using quantitative reverse transcription-polymerase chain reaction, in situ hybridization (ISH), and immunohistochemistry (IHC), we have confirmed specific expression in the E15.5 subplate for 13 selected genes, which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c, and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to synapse formation and axonal growth and guidance in subplate cells. PMID:21862448

  19. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed Central

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-01-01

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content. Images PMID:7524085

  20. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    PubMed

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis. PMID:27065589

  1. Developmental changes in DNA methylation and covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin gene promoters in Papio anubis.

    PubMed

    Lavelle, Donald; Vaitkus, Kestis; Hankewych, Maria; Singh, Mahipal; DeSimone, Joseph

    2006-01-01

    The baboon is a suitable and relevant animal model to study the mechanism of human globin gene switching. This investigation addresses the role of DNA methylation and histone coding in globin gene switching in the baboon, Papio anubis. Bisulfite sequencing and chromatin immunoprecipitation studies were performed in erythroid cells purified from fetuses of varying gestational ages and from adult bone marrow to analyze the manner that changes in DNA methylation of the epsilon-, gamma-, and beta-globin promoters and association of ac-H3, ac-H4, H3-dimeK4, H3-dimeK36, and H3-dimeK79 with the epsilon-, gamma-, and beta-globin promoters occur during development. Changes in DNA methylation of the epsilon- and gamma-globin gene promoters during transitional stages of globin gene switching were consistent with the stochastic model of methylation and a role of DNA methylation in gene silencing. Enrichment of ac-H3, ac-H4, and pol II at the promoters of developmentally active genes was observed, while the pattern of distribution of H3-dimeK4 and H3-dimeK79 suggests that these modifications are found near both currently and formerly active promoters. Enrichment of H3-dimeK36 at the silenced epsilon-globin gene promoter was observed. These studies demonstrate that coordinated epigenetic modifications in the chromatin structure of the beta-like globin gene promoters accompany the highly regulated changes in expression patterns of these genes during development. PMID:16527500

  2. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules.

    PubMed

    Baù, Davide; Sanyal, Amartya; Lajoie, Bryan R; Capriotti, Emidio; Byron, Meg; Lawrence, Jeanne B; Dekker, Job; Marti-Renom, Marc A

    2011-01-01

    We developed a general approach that combines chromosome conformation capture carbon copy (5C) with the Integrated Modeling Platform (IMP) to generate high-resolution three-dimensional models of chromatin at the megabase scale. We applied this approach to the ENm008 domain on human chromosome 16, containing the α-globin locus, which is expressed in K562 cells and silenced in lymphoblastoid cells (GM12878). The models accurately reproduce the known looping interactions between the α-globin genes and their distal regulatory elements. Further, we find using our approach that the domain folds into a single globular conformation in GM12878 cells, whereas two globules are formed in K562 cells. The central cores of these globules are enriched for transcribed genes, whereas nontranscribed chromatin is more peripheral. We propose that globule formation represents a higher-order folding state related to clustering of transcribed genes around shared transcription machineries, as previously observed by microscopy. PMID:21131981

  3. Copy number polymorphism in the α-globin gene cluster of European rabbit (Oryctolagus cuniculus)

    PubMed Central

    Campos, R; Storz, J F; Ferrand, N

    2012-01-01

    Comparative genomic studies have revealed that mammals typically possess two or more tandemly duplicated copies of the α-globin (HBA) gene. The domestic rabbit represents an exception to this general rule, as this species was found to possess a single HBA gene. Previous electrophoretic surveys of HBA polymorphism in natural populations of the European rabbit (Oryctolagus cuniculus) revealed extensive geographic variation in the frequencies of three main electromorphs. The variation in frequency of two electromorphs is mainly partitioned between two distinct subspecies of European rabbit, and a third is restricted to the hybrid zone between the two rabbit subspecies in Iberia. Here we report the results of a survey of nucleotide polymorphism, which revealed HBA copy number polymorphism in Iberian populations of the European rabbit. By characterizing patterns of HBA polymorphism in populations from the native range of the European rabbit, we were able to identify the specific amino-acid substitutions that distinguish the previously characterized electromorphs. Within the hybrid zone, we observed the existence of a second HBA gene duplicate, named HBA2, that mostly represents a novel sequence haplotype, which occurs in higher frequency within the hybrid zone, and thus appears to have arisen in hybrids of the two distinct subspecies. Although this novel gene is also present in other wild Iberian populations, it is almost absent from French populations, which suggest a recent ancestry, associated with the establishment of the post-Pleistocene contact zone between the two European rabbit subspecies. PMID:22146981

  4. Gene expression dynamics during embryonic development in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The supply of maternal RNAs in fertilized egg and activation of embryonic genome during maternal-zygotic transition (MZT) are important for normal embryonic development. In order to identify genes and gene products that are essential in the regulation of embryonic development in rainbow trout, RNA-S...

  5. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

    PubMed

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  6. Embryonal brain tumors and developmental control genes

    SciTech Connect

    Aguzzi, A.

    1995-12-31

    Cell proliferation in embryogenesis and neoplastic transformation is thought to be controlled by similar sets of regulatory genes. This is certainly true for tumors of embryonic origin, such as Ewing sarcoma, Wilms` tumor and retinoblastoma, in which developmental control genes are either activated as oncogenes to promote proliferation, or are inactivated to eliminate their growth suppressing function. However, to date little is known about the genetic events underlying the pathogenesis of medulloblastoma, the most common brain tumor in children, which still carries an unfavourable prognosis. None of the common genetic alterations identified in other neuroectodermal tumors, such as mutation of the p53 gene or amplification of tyrosine kinase receptor genes, could be uncovered as key events in the formation of medulloblastoma. The identification of regulatory genes which are expressed in this pediatric brain tumor may provide an alternative approach to gain insight into the molecular aspects of tumor formation.

  7. Fetal Hemoglobin in Tunisian Sickle Cell Disease Patient: Relationship with Polymorphic Sequences Cis to the β-Globin Gene.

    PubMed

    Moumni, Imen; Ben Mustapha, Maha; Ben Mansour, Ikbel; Zoraï, Amine; Douzi, Kaïs; Sassi, Sarah; Chaouachi, Dorra; Mellouli, Fethi; Bejaoui, Mohamed; Abbes, Salem

    2016-03-01

    Fetal hemoglobin (HbF) plays a dominant role in ameliorating morbidity and mortality of hemoglobinopathies. We evaluated the effects of polymorphic markers within the β-globin gene cluster to identify the genetic mechanics that influence HbF on Tunisian sickling patients (n = 242). Haplotype analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the framework polymorphism was established by PCR-sequencing, four independent regions of interest were identified: the 5' region of β-LCR-HS2 site, the intervening sequence II (IVSII) region of two fetal (Gγ and Aγ) genes and the 5' region of β-globin gene. The correlation of these various Haplotypes and SNPs with HbF expression and clinical data was studied. Our data showed that among the various polymorphic markers analyzed, only the sequence (AT)xN12(AT)y in LCR HS2 region was significantly associated (p < 0.05) with increased HbF levels, suggesting that the β-globin gene cluster exerts a significant effect on HbF in sickle cell patients. This study can improve understanding of the physiopathology of the disease and aid to increase our ability to predict clinical severity. PMID:26855518

  8. Self-catalytic DNA depurination underlies human β-globin gene mutations at codon 6 that cause anemias and thalassemias.

    PubMed

    Alvarez-Dominguez, Juan R; Amosova, Olga; Fresco, Jacques R

    2013-04-19

    The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5'G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease. PMID:23457306

  9. Role of the GATA-1/FOG-1/NuRD Pathway in the Expression of Human β-Like Globin Genes▿

    PubMed Central

    Miccio, Annarita; Blobel, Gerd A.

    2010-01-01

    The human β-globin genes are expressed in a developmentally controlled fashion. Studies on the molecular mechanisms underlying the stage-specific regulation of globin genes have been fueled by the clinical benefit of elevated fetal γ-globin expression in patients with sickle cell anemia and thalassemia. Recent reports suggested a role of the hematopoietic transcription factor GATA-1, its cofactor FOG-1, and the associated chromatin remodeling complex NuRD in the developmental silencing of HBG1 and HBG2 gene expression. To examine whether FOG-1 via NuRD controls HBG1 and HBG2 silencing in vivo, we created mice in which the FOG-1/NuRD complex is disrupted (A. Miccio et al., EMBO J. 29:442-456, 2010) and crossed these with animals carrying the entire human β-globin gene locus as a transgene. We found that the FOG-1/NuRD interaction is dispensable for the silencing of human HBG1 and HBG2 expression. In addition, mutant animals displayed normal silencing of the endogenous embryonic globin genes. In contrast, a significant reduction of adult-type human and murine globin gene expression was found in adult bone marrows of mutant animals. These results suggest that, unexpectedly, NuRD is required for FOG-1-dependent activation of adult-type globin gene expression but is dispensable for human γ-globin silencing in vivo. PMID:20439494

  10. The Full Globin Repertoire of Turtles Provides Insights into Vertebrate Globin Evolution and Functions

    PubMed Central

    Schwarze, Kim; Singh, Abhilasha; Burmester, Thorsten

    2015-01-01

    Globins are small heme proteins that play an important role in oxygen supply, but may also have other functions. Globins offer a unique opportunity to study the functional evolution of genes and proteins. We have characterized the globin repertoire of two different turtle species: the Chinese softshell turtle (Pelodiscus sinensis) and the western painted turtle (Chrysemys picta bellii). In the genomes of both species, we have identified eight distinct globin types: hemoglobin (Hb), myoglobin, neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Therefore, along with the coelacanth, turtles are so far the only known vertebrates with a full globin repertoire. This fact allows for the first time a comparative analysis of the expression of all eight globins in a single species. Phylogenetic analysis showed an early divergence of neuroglobin and globin X before the radiation of vertebrates. Among the other globins, cytoglobin diverged first, and there is a close relationship between myoglobin and globin E; the position of globin Y is not resolved. The globin E gene was selectively lost in the green anole, and the genes coding for globin X and globin Y were deleted in chicken. Quantitative real-time reverse transcription polymerase chain reaction experiments revealed that myoglobin, neuroglobin, and globin E are highly expressed with tissue-specific patterns, which are in line with their roles in the oxidative metabolism of the striated muscles, the brain, and the retina, respectively. Histochemical analyses showed high levels of globin E in the pigment epithelium of the eye. Globin E probably has a myoglobin-like role in transporting O2 across the pigment epithelium to supply in the metabolically highly active retina. PMID:26078264

  11. A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.

    PubMed Central

    Gesto-Borroto, Reinier; Sánchez-Sánchez, Miriam; Arredondo-Peter, Raúl

    2015-01-01

    Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO) minus reduced) differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58. PMID:26594329

  12. Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin.

    PubMed

    Caplan, A; Kimura, T; Gould, H; Allan, J

    1987-01-01

    An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour. PMID:3586025

  13. The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

    PubMed Central

    Romero, Zulema; Campo-Fernandez, Beatriz; Wherley, Jennifer; Kaufman, Michael L; Urbinati, Fabrizia; Cooper, Aaron R; Hoban, Megan D; Baldwin, Kismet M; Lumaquin, Dianne; Wang, Xiaoyan; Senadheera, Shantha; Hollis, Roger P; Kohn, Donald B

    2015-01-01

    Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies. PMID:26029723

  14. Expression of a cellular gene cloned in herpes simplex virus: rabbit beta-globin is regulated as an early viral gene in infected fibroblasts.

    PubMed Central

    Smiley, J R; Smibert, C; Everett, R D

    1987-01-01

    We constructed nondefective herpes simplex virus type 1 recombinants bearing the intact rabbit beta-globin gene inserted into the viral gene for thymidine kinase to study the expression of a cellular gene when it is present in the viral genome during lytic viral infections. The globin promoter was activated to high levels during productive infection of Vero cells, giving rise to properly spliced and processed cytoplasmic globin transcripts. Expression of globin RNA occurred with early kinetics, was not affected by blocking viral DNA replication, and was strongly inhibited by preventing viral immediate-early protein synthesis with cycloheximide. These results support the hypothesis that temporal control of herpes simplex virus early gene expression is accomplished by mechanisms that are not restricted to viral promoters. In addition, these data show that a cellular transcript can be correctly processed and can accumulate to high levels during viral infection; this indicates that the mechanisms of virally induced shutoff of host RNA accumulation and degradation of host mRNAs do not depend on sequence-specific differentiation between host and viral RNAs. These findings also suggest that herpesviruses have considerable potential as high-capacity gene transfer vectors for a variety of applications. Images PMID:3037101

  15. alpha-Amanitin-insensitive transcription of mouse beta major-globin 5'-flanking and structural gene sequences correlates with mRNA expression.

    PubMed Central

    Carlson, D P; Ross, J

    1984-01-01

    A small proportion of the RNAs of mouse reticulocytes consists of beta major-globin mRNA sequences linked to sequences transcribed from the 5'-flanking region of the beta major-globin gene. These upstream RNAs are polyadenylylated and contain 700-800 nucleotides, and their 5' regions are heterogeneous. RNAs with similar or identical 5' regions are transcribed in cell-free extracts from a circular mouse beta major-globin gene template. Synthesis of most of the upstream RNAs in vitro is not inhibited by low levels (1 microgram/ml) of alpha-amanitin, indicating that they are transcribed by an enzyme(s) different from RNA polymerase II. During culture of mouse erythroleukemia cells with dimethyl sulfoxide, globin mRNA and upstream RNAs accumulate with similar kinetics. In contrast, upstream RNAs are not detected in hemin-treated cells. Images PMID:6595660

  16. Incidence of Alpha-Globin Gene Defect in the Lebanese Population: A Pilot Study

    PubMed Central

    Daher, Rose; Badra, Rebecca; el Rafei, Rym; Charafeddine, Lama

    2015-01-01

    Background. It is well established that the Mediterranean and Arab populations are at high risk for thalassemias in general and for alpha-thalassemia in particular. Yet, reports on alpha-thalassemia in Lebanon are still lacking. In this study, we aim at assessing the incidence of alpha-thalassemia in the Lebanese population. Methods. 230 newborns' dried blood cards remaining from routine neonatal screening at the American University of Beirut Medical Center were collected for DNA extraction. Samples were screened for the 21 most common α-globin deletions and point mutations reported worldwide, through multiplex Polymerase Chain Reaction (PCR) and Reverse-Hybridization technique. Results. Upon analyses, the carrier rate of α-thalassemia was found to be 8%. Two mutations detected the −α3,7 single gene deletion found in 75% of cases and the nongene deletion α2 IVS1 [−5nt] in the remaining samples. Conclusion. This study is the first dedicated to investigate α-thalassemia trait incidence in Lebanon. Data obtained demonstrates a high carrier rate in a relatively, highly consanguineous population; it also highlighted the presence of two common mutations. These results may be of an important impact on premarital and newborn screening policies in our country. PMID:25834820

  17. Recent advances in globin research using genome-wide association studies and gene editing.

    PubMed

    Orkin, Stuart H

    2016-03-01

    A long-sought goal in the hemoglobin field has been an improved understanding of the mechanisms that regulate the switch from fetal (HbF) to adult (HbA) hemoglobin during development. With such knowledge, the hope is that strategies for directed reactivation of HbF in adults could be devised as an approach to therapy for the β-hemoglobinopathies thalassemia and sickle cell disease. Recent genome-wide association studies (GWAS) led to identification of three loci (BCL11A, HBS1L-MYB, and the β-globin cluster itself) in which natural genetic variation is correlated with different HbF levels in populations. Here, the central role of BCL11A in control of HbF is reviewed from the perspective of how findings may be translated to gene therapy in the not-too-distant future. This summary traces the evolution of recent studies from the initial recognition of BCL11A through GWAS to identification of critical sequences in an enhancer required for its erythroid-specific expression, thereby highlighting an Achilles heel for genome editing. PMID:26866328

  18. Globin gene-associated restriction-fragment-length polymorphisms in southern African peoples.

    PubMed Central

    Ramsay, M; Jenkins, T

    1987-01-01

    The combination of polymorphic restriction-enzyme sites in the 3' region of the beta-globin gene cluster shows very little variation in southern-African Bantu-speaking black and Kalahari !Kung San populations. The sites of the 5' region, on the other hand, show marked variation, and two common haplotypes are present--the "Negro" type (- - - - +) and the "San" type (- + - - +)--in frequencies of .404 and .106, respectively, in the Bantu-speakers and .262 and .405, respectively, in the San. Twenty of 23 beta s-associated haplotypes in southern-African Bantu-speaking black subjects were the same as that found commonly in the Central African Republic (CAR)--i.e., the "Bantu" type--a finding providing the first convincing biological evidence for the common ancestry of geographically widely separated speakers of languages belonging to the Bantu family. The (-alpha) haplotype has a frequency of .21 in the Venda, .07 in both the Sotho-Tswana and the Nguni, and .06 among the !Kung San. These data are interpreted in the light of Plasmodium falciparum malaria selection and population movements in the African subcontinent. PMID:2891298

  19. β-globin gene transfer to human bone marrow for sickle cell disease

    PubMed Central

    Romero, Zulema; Urbinati, Fabrizia; Geiger, Sabine; Cooper, Aaron R.; Wherley, Jennifer; Kaufman, Michael L.; Hollis, Roger P.; Ruiz de Assin, Rafael; Senadheera, Shantha; Sahagian, Arineh; Jin, Xiangyang; Gellis, Alyse; Wang, Xiaoyan; Gjertson, David; DeOliveira, Satiro; Kempert, Pamela; Shupien, Sally; Abdel-Azim, Hisham; Walters, Mark C.; Meiselman, Herbert J.; Wenby, Rosalinda B.; Gruber, Theresa; Marder, Victor; Coates, Thomas D.; Kohn, Donald B.

    2013-01-01

    Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin–like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2–3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells. PMID:23863630

  20. High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation.

    PubMed

    Stees, Jared R; Hossain, Mir A; Sunose, Tomoki; Kudo, Yasushi; Pardo, Carolina E; Nabilsi, Nancy H; Darst, Russell P; Poudyal, Rosha; Igarashi, Kazuhiko; Huang, Suming; Kladde, Michael P; Bungert, Jörg

    2015-01-01

    Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation. PMID:26503787

  1. High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

    PubMed Central

    Stees, Jared R.; Hossain, Mir A.; Sunose, Tomoki; Kudo, Yasushi; Pardo, Carolina E.; Nabilsi, Nancy H.; Darst, Russell P.; Poudyal, Rosha; Igarashi, Kazuhiko; Kladde, Michael P.

    2015-01-01

    Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation. PMID:26503787

  2. Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells

    PubMed Central

    Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen; Mead, Paul E; Smeltzer, Matthew P; Weiss, Mitchell J; Wilber, Andrew; Persons, Derek A

    2015-01-01

    Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans. PMID:26665131

  3. RNA sequencing for increasing gene discovery and and coverage using globin RNA reduced porcine blood samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Transcriptome analysis in porcine whole blood will provide major insights to decipher genetic mechanisms for host responses to viral infection. The abundance of porcine globin transcripts, however, impedes the ability to detect less abundant transcripts. The objective of our study was to...

  4. Dynamics of α-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34+ cells in culture

    PubMed Central

    Mahajan, Milind C; Karmakar, Subhradip; Krause, Diane; Weissman, Sherman M

    2009-01-01

    Objective The aim of the present study has been to establish serum free culture conditions for the ex vivo expansion and differentiation of human CD34+ cells into erythroid lineage and to study the chromatin structure, gene expression and transcription factor recruitment at the α–globin locus in the developing erythron. Methods A basal IMDM cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for the expansion and differentiation of the CD34+ cells. Expression patterns of the alpha and beta like genes at various stages of erythropoiesis was studied by Reverse transcriptase (RT)-qPCR analysis, profile of key erythroid transcription factors was investigated by western blotting, and the chromatin structure and transcription factor recruitment at the alpha globin locus was investigated by ChIP-qPCR analysis. Results Human CD34+ cells in the serum free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the α-globin locus during erythroid differentiation of CD34+ cells. NF-E2 was present at upstream activator sites even before addition of erythropoietin (Epo), while bound GATA-1 was only detectable after Epo treatment. After seven days of erythropoietin treatment, H3K4Me2 modification uniformly increases throughout the α–globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2 and GATA-1 were restricted to certain HS sites of the enhancer and theta gene, and were conspicuously low at the α-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the α-globin locus as CD34+ cells differentiated into erythroid pathway. Conclusion Our results indicate that remodeling of the upstream elements may be the primary event in activation of α–globin gene expression. Activation of

  5. Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts

    PubMed Central

    Vinjamur, Divya S.; Alhashem, Yousef N.; Mohamad, Safa F.; Amin, Parth; Williams, David C.; Lloyd, Joyce A.

    2016-01-01

    In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies. PMID:26840243

  6. Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts.

    PubMed

    Vinjamur, Divya S; Alhashem, Yousef N; Mohamad, Safa F; Amin, Parth; Williams, David C; Lloyd, Joyce A

    2016-01-01

    In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies. PMID:26840243

  7. Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

    PubMed Central

    Bruno, J; Reich, N; Lucas, J J

    1981-01-01

    The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed. Images PMID:7346715

  8. A novel δ-globin gene mutation (HBD: c.323G>A) masking the diagnosis of β-thalassemia: a first report from India.

    PubMed

    Jain, Sachin; Edison, Eunice S; Mathews, Vikram; Shaji, R V

    2012-05-01

    An elevated HbA(2) (α2δ2) level (>3.5%) is a well-established diagnostic test for heterozygous β-thalassemia. Mutations in the δ-globin gene can cause decreased expression of HbA(2), resulting in heterozygous β-thalassemia with normal levels of HbA(2). In this report, we describe a novel missense mutation in δ-globin (HBD: c.323G>A, Gly > Asp) in an Indian family with heterozygous β-thalassemia with normal HbA(2) levels. PMID:22477537

  9. A New δ-Globin Gene Variant: Hb A2-Fengshun [δ121(GH4)Glu→Lys (HBD: c.364G > A)].

    PubMed

    Yan, Jin-Mei; Zhou, Jian-Ying; Xie, Xing-Mei; Li, Jian; Li, Dong-Zhi

    2016-06-01

    An elevated Hb A2 (α2δ2 level) is a diagnostic marker for heterozygous β-thalassemia (β-thal). Mutations in the δ-globin gene can cause decreased expression of Hb A2, compromising screening for heterozygous β-thal. In this report, we describe a novel missense mutation of the δ-globin [Hb A2-Fengshun or δ121(GH4)Glu→Lys, HBD: c.364G > A] in a Chinese individual who had coinherited a heterozygous β-thal with a normal Hb A2 level. PMID:27117573

  10. A novel deletion/insertion caused by a replication error in the β-globin gene locus control region.

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Meley, Roland; Pondarré, Corinne; Francina, Alain

    2011-01-01

    Deletions in the β-globin locus control region (β-LCR) lead to (εγδβ)(0)-thalassemia [(εγδβ)(0)-thal]. In patients suffering from these rare deletions, a normal hemoglobin (Hb), phenotype is found, contrasting with a hematological thalassemic phenotype. Multiplex-ligation probe amplification (MLPA) is an efficient tool to detect β-LCR deletions combined with long-range polymerase chain reaction (PCR) and DNA sequencing to pinpoint deletion breakpoints. We present here a novel 11,155 bp β-LCR deletion found in a French Caucasian patient which removes DNase I hypersensitive site 2 (HS2) to HS4 of the β-LCR. Interestingly, a 197 bp insertion of two inverted sequences issued from the HS2-HS3 inter-region is present and suggests a complex rearrangement during replication. Carriers of this type of thalassemia can be misdiagnosed as an α-thal trait. Consequently, a complete α- and β-globin gene cluster analysis is required to prevent a potentially damaging misdiagnosis in genetic counselling. PMID:21797698

  11. A new Frameshift mutation on the α2-globin gene causing α⁺-thalassemia: codon 43 (TTC>-TC or TTC>T-C).

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Barro, Claire; Francina, Alain

    2012-01-01

    We report a new mutation on the α2-globin gene causing α(+)-thalassemia (α(+)-thal) with a deletion of a single nucleotide (T) at amino acid residue 43 [HBA2:c.130delT or HBA2:c.131delT]. This frameshift deletion gives rise to a premature termination codon at codon 47. PMID:22738776

  12. Heterozygosis deficit of polymorphic markers linked to the β-globin gene cluster region in the Iranian population

    PubMed Central

    Moradi, Tahereh; Vallian, Reihaneh; Fazeli, Zahra; Haghighatnia, Asieh; Vallian, Sadeq

    2015-01-01

    Objective(s): Iran is considered as one of the high-prevalence areas for β-thalassemia with a rate of about 10% carrier frequency. Molecular diagnosis of the disease is performed both by direct sequencing and indirectly by the use of polymorphic markers present in the beta globin gene cluster. However, to date there is no reliable information on the application of the markers in the Iranian population. Here we report the results of an extended molecular analysis of five RFLP markers, XmnI, HindIIIA, HindIIIG, RsaI and HinfI, located within the β-globin gene cluster region in four subpopulations of Iran. Materials and Methods: A total of 552 blood samples taken from the Iranian subpopulations including Isfahan, Chaharmahal-O-Bakhtiari, Khuzestan and Hormozgan were genotyped using PCR-RFLP and sequencing. The allele frequency, the expected and observed heterozygosity, and Shannon’s information index (I) of these markers were calculated. Results: Distribution of the allele frequencies for XmnI, HindIIIA, HindIIIG, RsaI and HinfI polymorphic markers did not differ significantly among the subpopulations examined. Overall observed heterozygosity ranged from 0.1706 for HindIIIA to 0.4484 for RsaI. The Shannon index was <1 for all the polymorphic markers in the populations studied. The data indicated that heterozygosity of these markers was low in the Iranian population. Conclusion: The results suggested that genotyping of these markers is not informative enough once used as single markers for prenatal diagnosis and carrier detection of β-thalassemia in the Iranian population. However, haplotyping of these markers may provide more useful data in linkage analysis and prenatal diagnosis as well as carrier detections for β-thalassemia in Iranians. PMID:26229579

  13. Characterization of a globin-coupled oxygen sensor with a gene-regulating function.

    PubMed

    Thijs, Liesbet; Vinck, Evi; Bolli, Alessandro; Trandafir, Florin; Wan, Xuehua; Hoogewijs, David; Coletta, Massimiliano; Fago, Angela; Weber, Roy E; Van Doorslaer, Sabine; Ascenzi, Paolo; Alam, Maqsudul; Moens, Luc; Dewilde, Sylvia

    2007-12-28

    Globin-coupled sensors (GCSs) are multiple-domain transducers, consisting of a regulatory globin-like heme-binding domain and a linked transducer domain(s). GCSs have been described in both Archaea and bacteria. They are generally assumed to bind O(2) (and perhaps other gaseous ligands) and to transmit a conformational change signal through the transducer domain in response to fluctuating O(2) levels. In this study, the heme-binding domain, AvGReg178, and the full protein, AvGReg of the Azotobacter vinelandii GCS, were cloned, expressed, and purified. After purification, the heme iron of AvGReg178 was found to bind O(2). This form was stable over many hours. In contrast, the predominant presence of a bis-histidine coordinate heme in ferric AvGReg was revealed. Differences in the heme pocket structure were also observed for the deoxygenated ferrous state of these proteins. The spectra showed that the deoxygenated ferrous derivatives of AvGReg178 and AvGReg are characterized by a penta-coordinate and hexa-coordinate heme iron, respectively. O(2) binding isotherms indicate that AvGReg178 and AvGReg show a high affinity for O(2) with P(50) values at 20 degrees C of 0.04 and 0.15 torr, respectively. Kinetics of CO binding indicate that AvGReg178 carbonylation conforms to a monophasic process, comparable with that of myoglobin, whereas AvGReg carbonylation conforms to a three-phasic reaction, as observed for several proteins with bis-histidine heme iron coordination. Besides sensing ligands, in vitro data suggest that AvGReg(178) may have a role in O(2)-mediated NO-detoxification, yielding metAvGReg(178) and nitrate. PMID:17925395

  14. Regulation of Gγ-Globin Gene by ATF2 and Its Associated Proteins through the cAMP-Response Element

    PubMed Central

    Dhar, Ruby; Mahajan, Milind; Choudhury, Alina; Weissman, Sherman; Pace, Betty S.

    2013-01-01

    The upstream Gγ-globin cAMP-response element (G-CRE) plays an important role in regulating Gγ-globin expression through binding of ATF2 and its DNA-binding partners defined in this study. ATF2 knockdown resulted in a significant reduction of γ-globin expression accompanied by decreased ATF2 binding to the G-CRE. By contrast, stable ATF2 expression in K562 cells increased γ-globin transcription which was reduced by ATF2 knockdown. Moreover, a similar effect of ATF2 on γ-globin expression was observed in primary erythroid progenitors. To understand the role of ATF2 in γ-globin expression, chromatographically purified G-CRE/ATF2-interacting proteins were subjected to mass spectrometry analysis; major binding partners included CREB1, cJun, Brg1, and histone deacetylases among others. Immunoprecipitation assays demonstrated interaction of these proteins with ATF2 and in vivo GCRE binding in CD34+ cells undergoing erythroid differentiation which was correlated with γ-globin expression during development. These results suggest synergism between developmental stage-specific recruitments of the ATF2 protein complex and expression of γ-globin during erythropoiesis. Microarray studies in K562 cells support ATF2 plays diverse roles in hematopoiesis and chromatin remodeling. PMID:24223142

  15. Two novel copy number variations involving the α-globin gene cluster on chromosome 16 cause thalassemia in two Chinese families.

    PubMed

    Hu, Lingling; Shang, Xuan; Yi, Sheng; Cai, Ren; Li, Zhetao; Liu, Cuixian; Liang, Yidan; Cai, Decheng; Zhang, Feng; Xu, Xiangmin

    2016-06-01

    Copy number variations (CNVs) can cause many genetic disorders and the structure analysis of unknown CNVs is important for clinical diagnosis. The human α-globin gene cluster lies close to the telomere of the short arm on chromosome 16. Copy number variations of this region produce excessive or insufficient α-globin chains which imbalances the β-globin chains, resulting in thalassemia. However, these CNVs usually cannot be precisely defined by traditional methods. Here, we designed a technique strategy and applied it to identify two CNVs involving the α-globin gene cluster causing thalassemia in two Chinese families. A novel 282 kb duplication (αααα(282)) was identified in family A and a novel 235 kb deletion (--(235)) in family B. Proband A is a coinheritance of β(CD41-42) and αααα(282) and showed severe β-thalassemia intermedia phenotype. Proband B is a compound heterozygote of --(235)/α(CS)α genotype and was diagnosed with hemoglobin H disease. The clinical phenotypic features of the CNVs carriers were described, together with a complete picture of molecular structure of these rearrangements. Two CNVs are novel rearrangements in α-globin clusters and the αααα(282) is the first to identify the exact insert position of a duplication region from the telomere on chromosome 16. In a conclusion, successful identification and characterization of these two novel CNVs not only demonstrates the precision and effectiveness of our strategy in analyzing the structure of unknown CNVs, but also extended the spectrum of thalassemia and provide new examples for studying genomic recombination. PMID:27000657

  16. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    PubMed Central

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  17. Specific repression of β-globin promoter activity by nuclear ferritin

    PubMed Central

    Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

    2001-01-01

    Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

  18. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

    PubMed

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  19. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations

    PubMed Central

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease’s high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics’ assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions’ setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  20. Gene targeting in embryonic stem cells, II: conditional technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  1. Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes

    PubMed Central

    Russell, Meaghan K.; Longoni, Mauro; Wells, Julie; Maalouf, Faouzi I.; Tracy, Adam A.; Loscertales, Maria; Ackerman, Kate G.; Pober, Barbara R.; Lage, Kasper; Bult, Carol J.; Donahoe, Patricia K.

    2012-01-01

    Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre–B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies. PMID:22315423

  2. Sickle cell disorder, beta-globin gene cluster haplotypes and alpha-thalassemia in neonates and adults from Guadeloupe.

    PubMed

    Kéclard, L; Romana, M; Lavocat, E; Saint-Martin, C; Berchel, C; Mérault, G

    1997-05-01

    We have studied haplotype of beta(S) chromosome and alpha-globin gene status in 534 patients (255 adults and 279 children of whom 159 neonates) from Guadeloupe with various sickle cell-related conditions, namely SS (n = 298), SC (n = 170), S-beta-thal (n = 56), and other rare forms (n = 10). Haplotype data on beta(S) chromosomes confirm our previous observation that Benin type is the most prevalent (75%) beta(S) chromosome in Guadeloupe, in disagreement with the historical records. Comparison of the frequency of distribution of various beta(S) haplotypes between neonates and adults on the one hand and between SS and SC cases on the other shows that the current beta(S) haplotype distribution in this island is not distorted by haplotype-related differential survival. We also show that the frequency of alpha-thalassemia (-3.7 kb) in Guadeloupe is one of the highest recorded in this region involved in Atlantic slave trade and also failed to reveal any age-dependent increase in frequency. We conclude that the African component of Guadeloupe is distinct from that of Brazil and Cuba but is close to that of Jamaica. PMID:9136913

  3. Developmental regulation of embryonic genes in plants

    SciTech Connect

    Borkird, C.; Choi, Jung, H.; Jin, Zhenghua; Franz, G.; Hatzopoulos, P.; Chorneaus, R.; Bonas, U.; Pelegri, F.; Sung, Z.R.

    1988-09-01

    Somatic embryogenesis from cultured carrot cells progresses through successive morphogenetic stages termed globular, heart, and torpedo. To understand the molecular mechanisms underlying plant embryogenesis, the authors isolated two genes differentially expressed during embryo development. The expression of these two genes is associated with heart-stage embryogenesis. By altering the culture conditions and examining their expressions in a developmental variant cell line, they found that these genes were controlled by the developmental program of embryogenesis and were not directly regulated by 2,4-dichlorophenoxyacetic acid, the growth regulator that promotes unorganized growth of cultured cells and suppresses embryo morphogenesis. These genes are also expressed in carrot zygotic embryos but not in seedlings or mature plants.

  4. Differential regulation of the α-globin locus by Krüppel-like factor 3 in erythroid and non-erythroid cells

    PubMed Central

    2014-01-01

    Background Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. Results We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3−/− mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3 −/− erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. Conclusions These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression. PMID:24885809

  5. The phylogenetic history of New World monkey beta globin reveals a platyrrhine beta to delta gene conversion in the atelid ancestry.

    PubMed

    Prychitko, Tom; Johnson, Robert M; Wildman, Derek E; Gumucio, Deborah; Goodman, Morris

    2005-04-01

    Orthologues of the beta globin gene locus from 10 New World monkey species were sequenced and aligned against available beta and delta globin sequences from rabbit and other primates. Where needed, additional primate sequencing was performed. Phylogenetic analysis identified a beta to delta conversion in the stem of the Anthropoidea, stretching from the 3' part of the proximal promotor to the 5' start of intron 2, consistent with earlier findings. No further conversion appeared to have occurred in the descent of the catarrhines. Within the New World monkey lineage that led to spider monkey and other atelids, another shorter gene conversion was found, spanning adjacent parts of exon 1 and intron 1. The analysis also confirmed that galago beta had replaced galago delta, that an earlier loriform-specific gene conversion extended over intron 2, and that gene conversion throughout the main gene conversion region occurred in the tarsiiform lineage. Platyrrhine phylogenetic relationships were investigated with beta sequences restricted to those that were not involved in gene conversions. This phylogeny generally agreed with results from other nuclear genes. The one exception was that the beta sequences did not place the callitrichine clade within the Cebidae but weakly joined the callitrichine and atelid clades. PMID:15737593

  6. Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice

    PubMed Central

    Costa, Flávia C.; Fedosyuk, Halyna; Chazelle, Allen M.; Neades, Renee Y.; Peterson, Kenneth R.

    2012-01-01

    Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. PMID:23284307

  7. Identification of Mechanosensitive Genes during Embryonic Bone Formation

    PubMed Central

    Nowlan, Niamh C.; Prendergast, Patrick J.; Murphy, Paula

    2008-01-01

    Although it is known that mechanical forces are needed for normal bone development, the current understanding of how biophysical stimuli are interpreted by and integrated with genetic regulatory mechanisms is limited. Mechanical forces are thought to be mediated in cells by “mechanosensitive” genes, but it is a challenge to demonstrate that the genetic regulation of the biological system is dependant on particular mechanical forces in vivo. We propose a new means of selecting candidate mechanosensitive genes by comparing in vivo gene expression patterns with patterns of biophysical stimuli, computed using finite element analysis. In this study, finite element analyses of the avian embryonic limb were performed using anatomically realistic rudiment and muscle morphologies, and patterns of biophysical stimuli were compared with the expression patterns of four candidate mechanosensitive genes integral to bone development. The expression patterns of two genes, Collagen X (ColX) and Indian hedgehog (Ihh), were shown to colocalise with biophysical stimuli induced by embryonic muscle contractions, identifying them as potentially being involved in the mechanoregulation of bone formation. An altered mechanical environment was induced in the embryonic chick, where a neuromuscular blocking agent was administered in ovo to modify skeletal muscle contractions. Finite element analyses predicted dramatic changes in levels and patterns of biophysical stimuli, and a number of immobilised specimens exhibited differences in ColX and Ihh expression. The results obtained indicate that computationally derived patterns of biophysical stimuli can be used to inform a directed search for genes that may play a mechanoregulatory role in particular in vivo events or processes. Furthermore, the experimental data demonstrate that ColX and Ihh are involved in mechanoregulatory pathways and may be key mediators in translating information from the mechanical environment to the molecular

  8. Thalidomide is more efficient than sodium butyrate in enhancing GATA-1 and EKLF gene expression in erythroid progenitors derived from HSCs with β-globin gene mutation

    PubMed Central

    Jalali Far, Mohammad Ali; Dehghani Fard, Ali; Hajizamani, Saiedeh; Mossahebi-Mohammadi, Majid; Yaghooti, Hamid; Saki, Najmaldin

    2016-01-01

    Background: Efficient induction of fetal hemoglobin (HbF) is considered as an effective therapeutic approach in beta thalassemia. HbF inducer agents can induce the expression of γ-globin gene and produce high levels of HbF via different epigenetic and molecular mechanisms. Thalidomide and sodium butyrate are known as HbF inducer drugs. Material and methods: CD133+ stem cells were isolated from umbilical cord blood of a newborn with minor β-thalassemia in order to evaluate the effects of these two drugs on the in vitro expression of GATA-1 and EKLF genes as erythroid transcription factors. CD133+ stem cells were expanded and differentiated into erythroid lineage and then treated with thalidomide and sodium butyrate and finally analyzed by quantitative real-time PCR. Statistical analysis was performed using student’s t-test by SPSS software. Results: Thalidomide and sodium butyrate increased GATA-1 and EKLF gene expression, compared to the non-treated control (P<0.05). Conclusion: Thalidomide was more efficient than sodium butyrate in augmenting expression of GATA-1 and EKLF genes. It seems that GATA-1 and EKLF have crucial roles in the efficient induction of HbF by thalidomide. PMID:27047649

  9. A novel β-globin gene mutation HBB.c.22 G>C produces a hemoglobin variant (Hb Vellore) mimicking HbS in HPLC.

    PubMed

    Edison, E S; Sathya, M; Rajkumar, S V; Nair, S C; Srivastava, A; Shaji, R V

    2012-10-01

    Hemoglobinopathies are highly prevalent in Indian population. DNA analysis to detect causative mutations is required for identifying rare hemoglobin variants or when hematological results are discordant with the clinical phenotype. In this report, we describe a novel hemoglobin variant caused by a mutation in beta-globin gene, Codon 7 GAG→CAG (Glu→Gln) that elutes in the position of sickle haemoglobin (HbS) in cation exchange high performance liquid chromatography. This report highlights possible diagnostic pitfalls in interpreting data solely based on haemoglobin analysis and usefulness of mutation screening in definitive diagnosis of hemoglobinopathies. PMID:22471768

  10. Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

    PubMed Central

    Byrnes, Colleen; Kaushal, Megha; Rabel, Antoinette; Tumburu, Laxminath; Allwardt, Joshua M.; Miller, Jeffery L.

    2015-01-01

    Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30–40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts. PMID:26675483

  11. An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter.

    PubMed

    Lewis, C D; Clark, S P; Felsenfeld, G; Gould, H

    1988-07-01

    The promoter region of the chicken adult beta-globin gene contains a sequence of 16 deoxyguanosine residues located at a nucleosome boundary in tissues where the gene is inactive. In definitive erythrocytes that express the beta-globin gene, the nucleosome is displaced, the G-string and adjacent sequences are occupied by sequence-specific DNA-binding proteins, and a nuclease hypersensitive domain is generated in this region. To gain insight into the role of the G-string in this series of events, we have examined the proteins that bind to it. Using the gel mobility shift assay and a monoclonal antibody that blocks specific binding to the G-string, we have identified a specific protein, BGP1, that is found only in chicken erythroid cells and appears at the same time, or shortly before, the changes in chromatin structure. The antibody interacts strongly with BGP1 and cross-reacts weakly with Sp1. Although both BGP1 and Sp1 require Zn2+ for their DNA-binding activity, these proteins differ in their binding-site specificities, chromatographic properties, and molecular weights. In contrast to Sp1, which is found in a wide variety of cell types, BGP1 is restricted to erythrocytes and is most abundant in definitive erythrocytes. Thus, its presence corresponds to the tissue- and stage-specific occupancy of the G-string in vivo. PMID:3209071

  12. Molecular and functional characterization of broccoli EMBRYONIC FLOWER 2 genes.

    PubMed

    Liu, Mao-Sen; Chen, Long-Fang O; Lin, Chun-Hung; Lai, Ying-Mi; Huang, Jia-Yuan; Sung, Zinmay Renee

    2012-07-01

    Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development. PMID:22537758

  13. Spatiotemporal control of embryonic gene expression using caged morpholinos.

    PubMed

    Shestopalov, Ilya A; Chen, James K

    2011-01-01

    Embryonic development depends on spatial and temporal control of gene function, and deciphering the molecular mechanisms that underlie pattern formation requires methods for perturbing gene expression with similar precision. Emerging chemical technologies can enable such perturbations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to photo-inactivate genes in zebrafish embryos with spatiotemporal control. This chapter describes general principles for cMO design and methods for cMO assembly in three steps from commercially available reagents. Experimental techniques for the microinjection and photoactivation of these reagents are described in detail, as well as the preparation and application of caged fluorescein dextran (cFD) for labeling irradiated cells. Using these protocols, cMOs can be effective tools for functional genomic studies in zebrafish and other model organisms. PMID:21924162

  14. A Phylogenetic Analysis of the Globins in Fungi

    PubMed Central

    Hoogewijs, David; Dewilde, Sylvia; Vierstraete, Andy; Moens, Luc; Vinogradov, Serge N.

    2012-01-01

    Background All globins belong to one of three families: the F (flavohemoglobin) and S (sensor) families that exhibit the canonical 3/3 α-helical fold, and the T (truncated 3/3 fold) globins characterized by a shortened 2/2 α-helical fold. All eukaryote 3/3 hemoglobins are related to the bacterial single domain F globins. It is known that Fungi contain flavohemoglobins and single domain S globins. Our aims are to provide a census of fungal globins and to examine their relationships to bacterial globins. Results Examination of 165 genomes revealed that globins are present in >90% of Ascomycota and ∼60% of Basidiomycota genomes. The S globins occur in Blastocladiomycota and Chytridiomycota in addition to the phyla that have FHbs. Unexpectedly, group 1 T globins were found in one Blastocladiomycota and one Chytridiomycota genome. Phylogenetic analyses were carried out on the fungal globins, alone and aligned with representative bacterial globins. The Saccharomycetes and Sordariomycetes with two FHbs form two widely divergent clusters separated by the remaining fungal sequences. One of the Saccharomycete groups represents a new subfamily of FHbs, comprising a previously unknown N-terminal and a FHb missing the C-terminal moiety of its reductase domain. The two Saccharomycete groups also form two clusters in the presence of bacterial FHbs; the surrounding bacterial sequences are dominated by Proteobacteria and Bacilli (Firmicutes). The remaining fungal FHbs cluster with Proteobacteria and Actinobacteria. The Sgbs cluster separately from their bacterial counterparts, except for the intercalation of two Planctomycetes and a Proteobacterium between the Fungi incertae sedis and the Blastocladiomycota and Chytridiomycota. Conclusion Our results are compatible with a model of globin evolution put forward earlier, which proposed that eukaryote F, S and T globins originated via horizontal gene transfer of their bacterial counterparts to the eukaryote ancestor, resulting from

  15. The Prevalence and Molecular Spectrum of α- and β-Globin Gene Mutations in 14,332 Families of Guangdong Province, China

    PubMed Central

    Xu, Longchang; Wu, Li; Zhang, Liang; Ma, Yuanzhu; Chen, Tingting; Gao, Shuang; Liang, Juqing; Guo, Hao; Qin, Danqing; Wang, Jicheng; Yuan, Tenglong; Wang, Yixia; Huang, Wei-wei; He, Wen-Fei; Zhang, Yanxia; Liu, Chang; Xia, Sujian; Chen, Qingshan; Zhao, Qingguo; Zhang, Xiaozhuang

    2014-01-01

    Objective To reveal the familial prevalence and molecular variation of α- and β-globin gene mutations in Guangdong Province. Methods A total of 40,808 blood samples from 14,332 families were obtained and analyzed for both hematological and molecular parameters. Results A high prevalence of α- and β-globin gene mutations was found. Overall, 17.70% of pregnant women, 15.94% of their husbands, 16.03% of neonates, and 16.83% of couples (pregnant women and their husbands) were heterozygous carriers of α- or β-thalassemia. The regions with the highest prevalence were the mountainous and western regions, followed by the Pearl River Delta; the region with the lowest prevalence was Chaoshan. The total familial carrier rate (both spouses were α- or β-thalassemia carriers) was 1.87%, and the individual carrier rates of α- and β-thalassemia were 1.68% and 0.20%, respectively. The total rate of moderate-to-severe fetal thalassemia was 12.78% among couples in which both parents were carriers. Conclusions There was a high prevalence of α- and β-thalassemia in Guangdong Province. This study will contribute to the development of thalassemia prevention and control strategies in Guangdong Province. PMID:24587075

  16. The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells.

    PubMed

    Golov, Arkadiy K; Gavrilov, Alexey A; Razin, Sergey V

    2015-01-01

    The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements. PMID:26436546

  17. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro.

    PubMed

    Srivastava, Anand S; Kaushal, Sharmeela; Mishra, Rangnath; Lane, Thomas A; Carrier, Ewa

    2006-07-28

    Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (alpha-globin, betaH-1 globin, beta-major globin, epsilon -globin, and zeta-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, epsilon-globin, gamma-globin, betaH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of

  18. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

    SciTech Connect

    Srivastava, Anand S.; Kaushal, Sharmeela; Mishra, Rangnath; Lane, Thomas A.; Carrier, Ewa . E-mail: assrivastava@ucsd.edu

    2006-07-28

    Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the

  19. Evolution of the primate beta-globin gene region: nucleotide sequence of the delta-beta-globin intergenic region of gorilla and phylogenetic relationships between African apes and man.

    PubMed

    Perrin-Pecontal, P; Gouy, M; Nigon, V M; Trabuchet, G

    1992-01-01

    A 6.0-kb DNA fragment from Gorilla gorilla including the 5' part of the beta-globin gene and about 4.5 kb of its upstream flanking region was cloned and sequenced. The sequence was compared to the human, chimpanzee, and macaque delta-beta intergenic region. This analysis reveals four tandemly repeated sequences (RS), at the same location in the four species, showing a variable number of repeats generating both intraspecific (polymorphism) and interspecific variability. These tandem arrays delimit five regions of unique sequence called IG for intergenic. The divergence for these IG sequences is 1.85 +/- 0.22% between human and gorilla, which is not significantly different from the value estimated in the same region between chimpanzee and human (1.62 +/- 0.21%). The CpG and TpA dinucleotides are avoided. CpGs evolve faster than other sequence sites but do not confuse phylogenetic inferences by producing parallel mutations in different lineages. About 75% of CpG doublets have become TpG or CpA since the common ancestor, in agreement with the methylation/deamination pattern. Comparison of this intergenic region gives information on branching order within Hominoidea. Parsimony and distance-based methods when applied to the delta-beta intergenic region provide evidence (although not statistically significant) that human and chimpanzee are more closely related to each other than to gorilla. CpG sites are indeed rich in information by carrying substitutions along the short internal branch. Combining these results with those on the psi eta-delta intergenic region, shows in a statistically significant way that chimpanzee is the closest relative of human. PMID:1556740

  20. Embryonic----Fetal Hb switch in humans: studies on erythroid bursts generated by embryonic progenitors from yolk sac and liver.

    PubMed

    Peschle, C; Migliaccio, A R; Migliaccio, G; Petrini, M; Calandrini, M; Russo, G; Mastroberardino, G; Presta, M; Gianni, A M; Comi, P

    1984-04-01

    The synthesis of embryonic (zeta, epsilon), fetal (alpha, gamma), and adult (beta) globin was evaluated in human yolk sacs (YS) and livers at different ontogenic stages (i.e., from 6 through 10-12 wk of age) by means of analytical isoelectric focusing. Globin production was comparatively evaluated in vivo (i.e., in directly labeled erythroblasts from YS and liver) and in vitro [i.e., in erythroid bursts generated in culture by erythroid burst-forming units (BFU-E) from the same erythropoietic tissues]. Erythroid bursts generated in vitro by BFU-E from 6-wk livers and YS show essentially a "fetal" globin synthetic pattern: this is in sharp contrast to the "embryonic" pattern in corresponding liver and YS erythroblasts directly labeled in vivo. The invitro phenomenon suggests that (i) 6-wk BFU-E constitute a new generation of progenitors, which have already switched from an embryonic to a fetal program, and/or (ii) expression of their fetal program is induced by unknown in vitro factor(s), which may underlie the in vivo switch at later ontogenic stages. It is emphasized that 6- to 7-wk BFU-E are endowed with the potential for in vitro synthesis of not only epsilon- and gamma-chains but also some beta-globin. In general, we observed an inverse correlation between the levels of epsilon- and beta-chain synthesis. These results, together with previous studies on fetal, perinatal, and adult BFU-E, are compatible with models suggesting that in ontogeny the chromatin configuration is gradually modified at the level of the non-alpha gene cluster, thus leading to a 5'----3' activation of globin genes in a balanced fashion. PMID:6201856

  1. Duplication and Divergence: The Evolution of Nematode Globins

    PubMed Central

    McNally, J.; Barris, W.; Blaxter, M. L.

    2009-01-01

    In common with many other groups, nematodes express globins with unknown functions. Nematode globin-like genes can be divided into class 1 globins, similar to vertebrate myoglobins, and a wide range of additional classes. Here we show that class 1 nematode globins possess a huge amount of diversity in gene sequence and structure. There is evidence for multiple events of gene duplication, intron insertion and loss between species, and for allelic variation effecting both synonymous and non-synonymous sites within species. We have also examined gene expression patterns in class I globins from a variety of species. The results show variation in the degree of gene expression, but the tissue specificity and temporal specificity of expression may be more conserved in the phylum. Because the structure-function relationships for the binding and transport of oxygen by globins are well understood, the consequences of genetic variation causing amino acid changes are explored. The gene family shows great promise for discovering unique insights into both structure-function relationships of globins and their physiologial roles. PMID:22661776

  2. A physiological delay in human fetal hemoglobin switching is associated with specific globin DNA hypomethylation.

    PubMed

    Perrine, S P; Greene, M F; Cohen, R A; Faller, D V

    1988-02-01

    The human fetal-to-adult globin switch normally occurs on a fixed schedule, beginning at 32-34 weeks gestation, and recent studies have suggested an association between this developmental inactivation of the fetal (gamma) globin genes and the appearance of methylation within and around these genes. We have studied a population of infants in whom this switch does not occur before birth (infants of diabetic mothers, IDM) and examined the patterns of methylation surrounding their active gamma-globin genes, in comparison to the gamma-globin genes of age-matched controls who have switched their pattern of globin gene expression on schedule. All genomic DNA samples from infants with delays in the globin switch demonstrated extensive hypomethylation in the region of the gamma-globin genes, comparable to that found in the genomes of fetuses of less than 21 weeks gestation. DNA from the erythroid cells of infants of 32-40 weeks gestation had no detectable hypomethylation in the gamma-globin region. These findings support the concept that hypomethylation is an accurate developmental marker of globin gene switching, and suggest that globin gene expression in IDM may be arrested at an early preswitch stage. PMID:2449361

  3. Prevalence of β(S)-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil.

    PubMed

    Shimauti, Eliana LitsukoTomimatsu; Silva, Danilo Grunig Humberto; de Souza, Eniuce Menezes; de Almeida, Eduardo Alves; Leal, Francismar Prestes; Bonini-Domingos, Claudia Regina

    2015-01-01

    The aim of this study was to determine the frequency of beta S-globin gene (β(S) globin) haplotypes and alpha thalassemia with 3.7 kb deletion (-α(3.7kb) thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of β(S) globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The β(S)globin haplotypes and -α(3.7kb) thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the -α(3.7kb) mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity. PMID:26500435

  4. Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.

    PubMed

    Wienert, Beeke; Funnell, Alister P W; Norton, Laura J; Pearson, Richard C M; Wilkinson-White, Lorna E; Lester, Krystal; Vadolas, Jim; Porteus, Matthew H; Matthews, Jacqueline M; Quinlan, Kate G R; Crossley, Merlin

    2015-01-01

    Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter. PMID:25971621

  5. Selection of Stable Reference Genes for Quantitative RT-PCR Comparisons of Mouse Embryonic and Extra-Embryonic Stem Cells

    PubMed Central

    Veazey, Kylee J.; Golding, Michael C.

    2011-01-01

    Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined. Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively. Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified m

  6. Characterization of Hb Calvino (HBB: c.406G > A): a new silent β-globin gene variant found in coexistence with α-thalassemia in a family of African origin.

    PubMed

    Marsella, Maria; Salvagno, Gianluca; Dolcini, Bernadetta; Ferlini, Alessandra; Ravani, Anna; Harteveld, Cornelis L; Giordano, Piero C; Borgna-Pignatti, Caterina

    2014-01-01

    We report a new silent β-globin gene variant found in a family from Angola living in the north eastern Italian city of Ferrara. The probands, two young sisters, presented with hematological parameters compatible with a β-thalassemia (β-thal) minor but with normal Hb A₂ levels and normal hemoglobin (Hb) separation on high performance liquid chromatography (HPLC). Molecular analyses revealed a homozygosity for the common -α(3.7) (rightward) deletion and heterozygosity for a novel transition (GCT > ACT) at codon 135 of the β-globin gene, leading to an Ala → Thr single amino acid substitution that was inherited from the healthy father. PMID:25222042

  7. Induction of gamma-globin gene transcription by hydroxycarbamide in primary erythroid cell cultures from Lepore patients.

    PubMed

    Calzolari, Roberta; Pecoraro, Alice; Borruso, Vito; Troia, Antonio; Acuto, Santina; Maggio, Aurelio; Di Marzo, Rosalba

    2008-05-01

    Increased expression of fetal haemoglobin (HbF) may ameliorate the clinical course of beta-thalassemia and sickle cell disease. Some pharmacological agents, such as hydroxycarbamide (HC), can increase fetal haemoglobin synthesis during adult life. Cellular selection and/or molecular mechanisms have been proposed to account for this increase. To explore the mechanism of action of HC we focused on homozygous Hb-Lepore patients that presented with high fetal haemoglobin levels and were good responders to HC treatment "in vivo". We performed primary erythroid cultures from peripheral blood of four homozygous Lepore patients. The increase in HBG (gamma-globin) transcription levels and HbF content in these cultures, after HC treatment, were detected by quantitative real time polymerase chain reaction analysis and flow cytometric analysis. Primary transcript "in-situ" hybridization analysis showed a 2-fold increase in the number of cells expressing both HBG alleles in HC-treated erythroid cultures. These studies, demonstrating the larger number of biallelic HBG expressing cells, suggest that HC is able to stimulate the activation of HBG transcription. These observations provide evidences that the molecular mechanism of action is involved in the increase of fetal haemoglobin production by HC. PMID:18422777

  8. Zebrafish globin Switching Occurs in Two Developmental Stages and is Controlled by the LCR

    PubMed Central

    Ganis, Jared J.; Hsia, Nelson; Trompouki, Eirini; de Jong, Jill L.O.; DiBiase, Anthony; Lambert, Janelle S.; Jia, Zhiying; Sabo, Peter J.; Weaver, Molly; Sandstrom, Richard; Stamatoyannopoulos, John A.; Zhou, Yi; Zon, Leonard I.

    2012-01-01

    Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5′-3′ to 3′-5′ orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching. PMID:22537494

  9. Detection of β-globin Gene Mutations Among β-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System–Polymerase Chain Reaction

    PubMed Central

    Hassan, Syahzuwan; Ahmad, Rahimah; Zakaria, Zubaidah; Zulkafli, Zefarina; Abdullah, Wan Zaidah

    2013-01-01

    Background: β-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of β-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming. Methods: A simple polymerase chain reaction (PCR) approach involving a multiplex amplification refractory mutation system (MARMS) and one amplification refractory mutation system (ARMS), consisting of 20 β-globin gene mutations, were designed and employed to investigate β-thalassaemia patients and carriers. Results: Out of 169 carriers tested with the MARMS, Cd 41/42 (–TTCT), Cd 26 (A–G) HbE, IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 78.1%. Among the Malays, Cd 26 (A–G) HbE, Cd 41/42 (–TTCT), IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 81.4%, whereas Cd 41/42 (–TTCT) and IVS 2–654 (C–T) were most common among the Chinese (79.1%). Conclusion: We propose the use of this cheap, easy to interpret, and simple system for the molecular diagnostics of β-thalassaemia among Malaysians at the Institute for Medical Research (IMR). PMID:23613656

  10. Characteristics of the beta-globin gene cluster haplotypes of three Han Chinese populations at Beijing, Xi'an, and Kunming as compared with those of other Asian populations.

    PubMed

    Shimizu, Koji; Nagaoka, Erika; Okada, Yusuke; Takeuchi, Yukiko; Harihara, Shinji; Omoto, Keiichi; Imanishi, Tadashi; Kim, Wook; Shin, Dong-Jik; Hao, Luping; Jin, Feng

    2008-10-01

    Haplotype frequencies of the beta-globin gene cluster of Han Chinese at Beijing, Xi'an, and Kunming were estimated, and their mutual genetic relationships were examined and compared to those of Buryats, Khalkhs, Evenkis, Oroqens, Koreans, and Colombian Amerindians. A major 5' subhaplotype (5' to the delta-globin gene), a major 3' subhaplotype (in and 3' to the beta-globin gene), and a major haplotype (combination of 5' and 3' subhaplotypes) are represented as + - - - -, - +, and + - - - - - +, respectively, and found in all three Han Chinese. A rare 5' subhaplotype, - - - - -, which is one of the possible ancestral types, was found only in Han Chinese at Kunming at low frequency (0.013), and a rare 3' subhaplotype, - -, was also observed in all three Han Chinese at low frequencies (0.009-0.014). The present haplotype frequency study suggested that the highest genetic affinity was found between Han Chinese at Beijing and those at Xi'an; the next highest was between Han Chinese at Beijing and Koreans, followed by that between Han Chinese at Beijing and Khalkhs, then that between Han Chinese at Xi'an and those at Kunming or Khalkhs, and finally that between Han Chinese at Beijing and those at Kunming. A genetic boundary between northern and southern Han Chinese was not evident in the present study. PMID:18553219

  11. Fannin-Lubbock-I [α₂β₂¹¹⁹(GLY>ASP)], a rare mutation in the beta-globin gene, has been detected for the first time in a Hindu Brahmin family in West Bengal, India.

    PubMed

    Basak, Jayasri; Bhattacharyya, Deboshree M; Mukhopadhyay, Ashis

    2014-06-01

    This study aims to describe the hemoglobin Fannin-Lubbock-I, which has a rare mutation substituting the amino acid glycine with aspartic acid at codon 119 of the β-globin chain. A Bengalee Hindu Brahmin family from Kolkata in West Bengal was the focus of this study. Molecular analysis using ARMS-PCR and direct DNA sequencing revealed the presence of a GGC > GAC mutation in codon 119 of the β-globin gene in a heterozygote state in three women of the same family. This is the first report of the hemoglobin Fannin-Lubbock-I from India. Our results will help to identify this mutation, which is relatively infrequent in our population. PMID:24802353

  12. Embryonic cerebrospinal fluid collaborates with the isthmic organizer to regulate mesencephalic gene expression.

    PubMed

    Parada, Carolina; Martín, Cristina; Alonso, María I; Moro, José A; Bueno, David; Gato, Angel

    2005-11-01

    Early in development, the behavior of neuroepithelial cells is controlled by several factors acting in a developmentally regulated manner. Recently it has been shown that diffusible factors contained within embryonic cerebrospinal fluid (CSF) promote neuroepithelial cell survival, proliferation, and neurogenesis in mesencephalic explants lacking any known organizing center. In this paper, we show that mesencephalic and mesencephalic+isthmic organizer explants cultured only with basal medium do not express the typically expressed mesencephalic or isthmic organizer genes analyzed (otx2 and fgf8, respectively) and that mesencephalic explants cultured with embryonic CSF-supplemented medium do effect such expression, although they exhibit an altered pattern of gene expression, including ectopic shh expression domains. Other trophic sources that are able to maintain normal neuroepithelial cell behavior, i.e., fibroblast growth factor-2, fail to activate this ectopic shh expression. Conversely, the expression pattern of the analyzed genes in mesencephalic+isthmic organizer explants cultured with embryonic cerebrospinal fluid-supplemented medium mimics the pattern for control embryos developed in ovo. We demonstrate that embryonic CSF collaborates with the isthmic organizer in regulation of the expression pattern of some characteristic neuroectodermal genes during early stages of central nervous system (CNS) development, and we suggest that this collaboration is not restricted to the maintenance of neuroepithelial cell survival. Data reported in this paper corroborate the hypothesis that factors contained within embryonic CSF contribute to the patterning of the CNS during early embryonic development. PMID:16180222

  13. Confirmation of the potential usefulness of two human beta globin pseudogene markers to estimate gene flows to and from sub-Saharan Africans.

    PubMed

    Ciminelli, Bianca Maria; Pompei, Fiorenza; Relucenti, Michela; Lum, J Koji; Simporé, Jacques; Spedini, Gabriella; Martínez-Labarga, Cristina; Pardo, Miguel G

    2002-04-01

    Two polymorphic sites, -107 and -100 with respect to the "cap" site of the human beta globin pseudogene, recently discovered in our laboratory, turned out to have an ethnically complementary distribution. The first site is polymorphic in Europeans, North Africans, Indians (Hindu), and Oriental Asians, and monomorphic in sub-Saharan Africans. Conversely, the second site is polymorphic in sub-Saharan African populations and monomorphic in the aforementioned populations. Here we report the gene frequencies of these two polymorphic sites in nine additional populations (Egyptians, Spaniards, Japanese, Chinese, Filipinos, Vietnamese, Africans from Togo and from Benin, and Pygmies), confirming their ethnospecificity and, through the analysis of these two markers in Oromo and Amhara of Ethiopia (two mixed populations), their usefulness in genetic admixture studies. Moreover, we studied another marker polymorphic in sub-Saharan African populations only, a TaqI restriction fragment length polymorphism located in the same region as the present markers, demonstrating the absence of linkage disequilibrium between it and the -100 site, so that we can exclude that the information they provide is redundant. PMID:12030652

  14. A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

    PubMed

    Renz, Peter F; Beyer, Tobias A

    2016-01-01

    Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA). PMID:25724912

  15. Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide.

    PubMed

    Shahid, Kazi Abdus; Majumdar, Alokes; Alam, Rowshon; Liu, Su-Ting; Kuan, Jean Y; Sui, Xuifen; Cuenoud, Bernard; Glazer, Peter M; Miller, Paul S; Seidman, Michael M

    2006-02-14

    Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A8) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A8 did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m) and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines. PMID:16460044

  16. Prevalence of βS-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil

    PubMed Central

    Shimauti, Eliana LitsukoTomimatsu; Silva, Danilo Grunig Humberto; de Souza, Eniuce Menezes; de Almeida, Eduardo Alves; Leal, Francismar Prestes; Bonini-Domingos, Claudia Regina

    2015-01-01

    The aim of this study was to determine the frequency of beta S-globin gene (βS globin) haplotypes and alpha thalassemia with 3.7 kb deletion (−α3.7kb thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of βS globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The βSglobin haplotypes and −α3.7kb thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the −α3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity. PMID:26500435

  17. Globin-coupled sensors, protoglobins, and the last universal common ancestor.

    PubMed

    Freitas, Tracey Allen K; Saito, Jennifer A; Hou, Shaobin; Alam, Maqsudul

    2005-01-01

    The strategy for detecting oxygen, carbon monoxide, nitric oxide, and sulfides is predominantly through heme-based sensors utilizing either a globin domain or a PAS domain. Whereas PAS domains bind various cofactors, globins bind only heme. Globin-coupled sensors (GCSs) were first described as regulators of the aerotactic responses in Bacillus subtilis and Halobacterium salinarum. GCSs were also identified in diverse microorganisms that appear to have roles in regulating gene expression. Functional and evolutionary analyses of the GCSs, their protoglobin ancestor, and their relationship to the last universal common ancestor (LUCA) are discussed in the context of globin-based signal transduction. PMID:15598488

  18. GLUT3 Gene Expression is Critical for Embryonic Growth, Brain Development and Survival

    PubMed Central

    Carayannopoulos, Mary O.; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U.

    2015-01-01

    Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. PMID:24529979

  19. Alpha-globin loci in homozygous beta-thalassemia intermedia.

    PubMed

    Triadou, P; Lapoumeroulie, C; Girot, R; Labie, D

    1983-01-01

    Homozygous beta-thalassemia intermediate (TI) differs from thalassemia major (TM) in being less severe clinically. Associated alpha-thalassemia could account for the TI phenotype by reducing the alpha/non-alpha chain imbalance. We have analyzed the alpha loci of 9 TI and 11 TM patients by restriction endonuclease mapping. All the TM and 7 of the TI patients have the normal complement of four alpha-globin genes (alpha alpha/alpha alpha). One TI patient has three alpha-globin genes (alpha alpha/-alpha), and another TI patient has five alpha genes (alpha alpha/alpha alpha alpha). PMID:6305827

  20. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.

    PubMed

    Montagner, Giulia; Gemmo, Chiara; Fabbri, Enrica; Manicardi, Alex; Accardo, Igea; Bianchi, Nicoletta; Finotti, Alessia; Breveglieri, Giulia; Salvatori, Francesca; Borgatti, Monica; Lampronti, Ilaria; Bresciani, Alberto; Altamura, Sergio; Corradini, Roberto; Gambari, Roberto

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  1. Characterization and embryonic expression of four amphioxus Frizzled genes with important functions during early embryogenesis.

    PubMed

    Qian, Guanghui; Li, Guang; Chen, Xiaoying; Wang, Yiquan

    2013-12-01

    The Wnt signaling pathway plays crucial roles in the embryonic patterning of all metazoans. Recent studies on Wnt genes in amphioxus have shed important insights into the evolution of the vertebrate Wnt gene family and their functions. Nevertheless, the potential roles of Wnt family receptors encoded by Frizzled (Fz) genes in amphioxus embryonic development remain to be investigated. In the present study, we identified four amphioxus Fz genes-AmphiFz1/2/7, AmphiFz4, AmphiFz5/8, and AmphiFz9/10-and analyzed their expression patterns during amphioxus embryogenesis. We found that these four Fz genes were maternally expressed and might be involved in early animal-vegetal axis establishment. The AmphiFz1/2/7 transcripts were detected in the central dorsal neural plate, mesoderm, the Hatschek's pit, and rim of the mouth, whereas those of AmphiFz4 were detected in the mesoderm, pharyngeal endoderm, and entire gut region. AmphiFz5/8 was exclusively expressed in the anterior-most region, whereas AmphiFz9/10 was expressed in the neural plate, somites, and tail bud. The dynamic and diverse expression patterns of amphioxus Fz genes suggest that these genes are not only associated with early embryonic axis establishment but also are involved in the development of several organs in amphioxus. PMID:24012522

  2. Hairpin-duplex equilibrium reflected in the A-->B transition in an undecamer quasi-palindrome present in the locus control region of the human beta-globin gene cluster.

    PubMed

    Kaushik, Mahima; Kukreti, Ritushree; Grover, Deepak; Brahmachari, Samir K; Kukreti, Shrikant

    2003-12-01

    Our recent work on an A-->G single nucleotide polymorphism (SNP) at the quasi-palindromic sequence d(TGGGG[A/G]CCCCA) of HS4 of the human beta-globin locus control region in an Indian population showed a significant association between the G allele and the occurrence of beta-thalassemia. Using UV-thermal denaturation, gel assay, circular dichroism (CD) and nuclease digestion experiments we have demonstrated that the undecamer quasi- palindromic sequence d(TGGGGACCCCA) (HPA11) and its reported polymorphic (SNP) version d(TGG GGGCCCCA) (HPG11) exist in hairpin-duplex equilibria. The biphasic nature of the melting profiles for both the oligonucleotides persisted at low as well as high salt concentrations. The HPG11 hairpin showed a higher T(m) than HPA11. The presence of unimolecular and bimolecular species was also shown by non-denaturating gel electrophoresis experiments. The CD spectra of both oligonucleotides showed features of the A- as well as B-type conformations and, moreover, exhibited a concentration dependence. The disappearance of the 265 nm positive CD signal in an oligomer concentration-dependent manner is indicative of an A-->B transition. The results give unprecedented insight into the in vitro structure of the quasi-palindromic sequence and provide the first report in which a hairpin-duplex equilibrium has been correlated with an A-->B interconversion of DNA. The nuclease-dependent degradation suggests that HPG11 is more resistant to nuclease than HPA11. Multiple sequence alignment of the HS4 region of the beta-globin gene cluster from different organisms revealed that this quasi-palindromic stretch is unique to Homo sapiens. We propose that quasi-palindromic sequences may form stable mini- hairpins or cruciforms in the HS4 region and might play a role in regulating beta-globin gene expression by affecting the binding of transcription factors. PMID:14627823

  3. Structural analysis of the 5 prime flanking region of the. beta. -globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa

    SciTech Connect

    Chebloune, Y.; Pagnier, J.; Trabuchet, G.; Faure, C.; Verdier, G.; Labie, D.; Nigon, V. )

    1988-06-01

    Haplotype analysis of the {beta}-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT){sub n} and (AT){sub x}T{sub y}, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT){sub n} and (AT){sub x}T{sub y} repeats. The authors found three additional structures for (AT){sub x}T{sub y} correlating with the geographic origin of the patients. Ten other nucleotide positions, 5{prime} and 3{prime} to the (AT){sub x}T{sub y} copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5{prime} flanking region of the {beta}-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  4. The gene for familial Mediterranean fever in both Armenians and non-Ashkenazi Jews is linked to the alpha-globin complex on 16p: evidence for locus homogeneity.

    PubMed Central

    Shohat, M; Bu, X; Shohat, T; Fischel-Ghodsian, N; Magal, N; Nakamura, Y; Schwabe, A D; Schlezinger, M; Danon, Y; Rotter, J I

    1992-01-01

    Familial Mediterranean fever (FMF) is a recurrent inflammatory disorder characterized by short episodes of fever, peritonitis, pleuritis, and arthritis. While FMF has been shown to be inherited in an autosomal recessive fashion in both non-Ashkenazi Jews and Armenian families, clinical differences have raised the possibility of genetic heterogeneity. As its pathogenesis is unknown, mapping of the gene for FMF may provide the first objective method for early and accurate diagnosis of this disease. After excluding 45% of the entire human genome, we studied 14 Armenian and 9 non-Ashkenazi Jewish families with FMF and tested linkage with the alpha-globin locus on chromosome 16. Analysis of the PvuII length polymorphism of the 3' HVR (hypervariable region) probe showed significant linkage with the FMF gene (maximum lod score [lodmax] = 9.76 at maximum recombination fraction [theta] = .076). In the Armenians, the lodmax = 3.61 at theta = .10; and for the non-Ashkenazi Jews, lodmax = 6.28 at theta = .06. There was no evidence for genetic heterogeneity between the Armenians and the non-Ashkenazi Jews (chi 2 = 1.28; P = .26) or within either ethnic group (chi 2 = .00; P = .50). Thus, the gene for FMF is linked to the alpha-globin complex on chromosome 16p in both non-Ashkenazi Jews and Armenians. PMID:1463015

  5. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  6. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    SciTech Connect

    Gao, Xiugong Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  7. The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability.

    PubMed

    Bhattacharya, Chitralekha; Aggarwal, Sita; Zhu, Rui; Kumar, Madhu; Zhao, Ming; Meistrich, Marvin L; Matin, Angabin

    2007-03-30

    Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain. PMID:17291453

  8. Surface functionalities of gold nanoparticles impact embryonic gene expression responses

    PubMed Central

    Truong, Lisa; Tilton, Susan C.; Zaikova, Tatiana; Richman, Erik; Waters, Katrina M.; Hutchison, James E.; Tanguay, Robert L.

    2012-01-01

    Incorporation of gold nanoparticles (AuNPs) into consumer products is increasing; however, there is a gap in available toxicological data to determine the safety of AuNPs. In this study, we utilised the embryonic zebrafish to investigate how surface functionalisation and charge influence molecular responses. Precisely engineered AuNPs with 1.5 nm cores were synthesised and functionalized with three ligands: 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol. Developmental assessments revealed differential biological responses when embryos were exposed to the functionalised AuNPs at the same concentration. Using inductively coupled plasma–mass spectrometry, AuNP uptake was confirmed in exposed embryos. Following exposure to MES- and TMAT-AuNPs from 6 to 24 or 6 to 48 h post fertilisation, pathways involved in inflammation and immune response were perturbed. Additionally, transport mechanisms were misregulated after exposure to TMAT and MES-AuNPs, demonstrating that surface functionalisation influences many molecular pathways. PMID:22263968

  9. Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

    PubMed Central

    Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

    2012-01-01

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

  10. Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the gamma-delta-beta-globin gene complex.

    PubMed Central

    Charache, S; Dover, G; Smith, K; Talbot, C C; Moyer, M; Boyer, S

    1983-01-01

    Increased production of fetal hemoglobin (HbF) was observed in a patient with sickle cell anemia treated with 5-azacytidine. Each of four courses of therapy resulted in a rapid and prolonged increase in the percentage of HbF containing reticulocytes (F reticulocytes) and HbF containing erythrocytes (F cells). The percentage of HbF in peripheral blood rose from 1.8 to 8.9%. The rise in HbF production was accompanied by an increase in peripheral blood hemoglobin concentration from 8 to 12 g/dl and an increase in mean erythrocyte volume. Treatment with 5-azacytidine resulted in hypomethylation of total genomic and a Y-chromosome-specific DNA fragment isolated from both peripheral blood and bone marrow. Of 15 restriction enzyme sites around the gamma-delta-beta-globin gene complex, only 2 became hypomethylated: one 107 bases 5' to the gamma G and the other 107 bases 5' to the gamma A globin genes. Images PMID:6192443

  11. Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anemia in the Atlantic and Pacific coastal regions of Colombia.

    PubMed

    Fong, Cristian; Lizarralde-Iragorri, María Alejandra; Rojas-Gallardo, Diana; Barreto, Guillermo

    2013-12-01

    Sickle cell anemia is a genetic disease with high prevalence in people of African descent. There are five typical haplotypes associated with this disease and the haplotypes associated with the beta-globin gene cluster have been used to establish the origin of African-descendant people in America. In this work, we determined the frequency and the origin of haplotypes associated with hemoglobin S in a sample of individuals with sickle cell anemia (HbSS) and sickle cell hemoglobin trait (HbAS) in coastal regions of Colombia. Blood samples from 71 HbAS and 79 HbSS individuals were obtained. Haplotypes were determined based on the presence of variable restriction sites within the β-globin gene cluster. On the Pacific coast of Colombia the most frequent haplotype was Benin, while on the Atlantic coast Bantu was marginally higher than Benin. Eight atypical haplotypes were observed on both coasts, being more diverse in the Atlantic than in the Pacific region. These results suggest a differential settlement of the coasts, dependent on where slaves were brought from, either from the Gulf of Guinea or from Angola, where the haplotype distributions are similar. Atypical haplotypes probably originated from point mutations that lost or gained a restriction site and/or by recombination events. PMID:24385850

  12. Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

    PubMed Central

    Soucie, Erinn L.; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J.-C.; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H.

    2016-01-01

    Differentiated macrophages can self-renew in tissues and expand long-term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. Single cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. PMID:26797145

  13. Differentiation of Murine Embryonic Stem Cells Induces Progesterone Receptor Gene Expression

    PubMed Central

    Sauter, Carley N.; McDermid, Rebecca L.; Weinberg, Amy L.; Greco, Tamara L.; Xu, Xiaojie; Murdoch, Fern E.; Fritsch, Michael K.

    2005-01-01

    The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERα, ERβ), are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen dependent manner by endogenous ERs or by transiently overexpressed ERα. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation PMID:16223481

  14. Locus assignment of alpha-globin structural mutations by hybrid-selected translation.

    PubMed Central

    Liebhaber, S A; Cash, F E

    1985-01-01

    The two human alpha-globin genes, alpha 1 and alpha 2 located 3.4 kilobases apart on chromosome 16, encode identical alpha-globin proteins. A mutation in either gene could result in a structural hemoglobinopathy. It has only recently become possible to assign an alpha-chain mutant to one of these two loci by using recombinant DNA technology. While definitive, this approach has necessitated the cloning and sequencing of the specific gene in question. We present an alternative approach which results in rapid and definitive assignment of an alpha-globin mutation to its encoding genetic locus. This approach uses the technique of hybrid-selected translation. Reticulocyte RNA from individuals with alpha-globin mutations can be fractionated into beta-, alpha 9 (total)-, alpha 1-, and alpha 2-globin mRNA by selective hybridization of each mRNA species to its respective complementary DNA (cDNA) immobilized on nitrocellulose paper. Each mRNA purified in this way can be translated in vitro, and the mRNA species (and hence gene locus) encoding the globin mutant can then be directly identified by gel analysis of the radiolabeled translation products. This procedure can be used to identify globin mutants as alpha or beta and to localize alpha-globin mutants to the alpha 1 or alpha 2 gene. We have used this technique to localize the two alpha-globin mutants, alpha 125Pro (Hb Quong Sze) and alpha 47HIS (Hb Hasharon), to the alpha 2 locus. This approach could potentially be expanded to serve as an alternative to peptide analysis for the initial characterization of all globin structural mutants. Images PMID:2981252

  15. A novel 26 bp deletion [HBB: c.20_45del26bp] in exon 1 of the β-globin gene causing β-thalassemia major.

    PubMed

    Edison, Eunice S; Venkatesan, Rajkumar S; Govindanattar, Sankari Devi; George, Biju; Shaji, Ramachandran V

    2012-01-01

    Molecular characterization of β-thalassemia (β-thal) is essential in prevention and in understanding the biology of the disease. Deletion mutations are relatively uncommon in β-thal. In this report, we describe a novel 26 bp deletion from codon 6 to codon 14 in the β-globin in a consanguineous family from Tamil Nadu, India. This novel mutation causes a shift in the normal reading frame of the β-globin coding sequence, and consequently, a premature chain termination of translation due to the creation of a stop codon at the position of codon 21. The identification of this novel deletional mutation adds to the repertoire of β-thal mutations in India. PMID:22233277

  16. Regulation of the retinal determination gene dachshund in the embryonic head and developing eye of Drosophila

    PubMed Central

    Anderson, Jason; Salzer, Claire L.; Kumar, Justin P.

    2009-01-01

    The retinal determination gene dachshund is distantly related to the family of Ski/Sno proto-oncogenes and influences the development of a wide range of tissues including the embryonic head, optic lobes, brain, central nervous system as well as the post-embryonic leg, wing, genital and eye-antennal discs. We were interested in the regulatory mechanisms that control the dynamic expression pattern of dachshund and in this report we set out to ascertain how the transcription of dachshund is modulated in the embryonic head and developing eye-antennal imaginal disc. We demonstrate that the TGFβ signaling cascade, the transcription factor zerknullt and several other patterning genes prevent dachshund from being expressed inappropriately within the embryonic head. Additionally, we show that several members of the eye specification cascade influence the transcription of dachshund during normal and ectopic eye development. Our results suggest that dachshund is regulated by a complex combinatorial code of transcription factors and signaling pathways. Unraveling this code may lead to an understanding of how dachshund regulates the development of many diverse tissue types including the eye. PMID:16780828

  17. Isolation and characterization of an avian slow myosin heavy chain gene expressed during embryonic skeletal muscle fiber formation.

    PubMed

    Nikovits, W; Wang, G F; Feldman, J L; Miller, J B; Wade, R; Nelson, L; Stockdale, F E

    1996-07-19

    We have isolated and begun characterization of the quail slow myosin heavy chain (MyHC) 3 gene, the first reported avian slow MyHC gene. Expression of slow MyHC 3 in skeletal muscle is restricted to the embryonic period of development, when the fiber pattern of future fast and slow muscle is established. In embryonic hindlimb development, slow MyHC 3 gene expression coincides with slow muscle fiber formation as distinguished by slow MyHC-specific antibody staining. In addition to expression in embryonic appendicular muscle, slow MyHC 3 is expressed continuously in the atria. Transfection of slow MyHC 3 promoter-reporter constructs into embryonic myoblasts that form slow MyHC-expressing fibers identified two regions regulating expression of this gene in skeletal muscle. The proximal promoter, containing potential muscle-specific regulatory motifs, permits expression of a reporter gene in embryonic slow muscle fibers, while a distal element, located greater than 2600 base pairs upstream, further enhances expression 3-fold. The slow muscle fiber-restricted expression of slow MyHC 3 during embryonic development, and expression of slow MyHC 3 promoter-reporter constructs in embryonic muscle fibers in vitro, makes this gene a useful marker to study the mechanism establishing the slow fiber lineage in the embryo. PMID:8663323

  18. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    PubMed

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. PMID:26006729

  19. Automated analysis of embryonic gene expression with cellular resolution in C. elegans

    PubMed Central

    Murray, John Isaac; Bao, Zhirong; Boyle, Thomas J.; Boeck, Max E.; Mericle, Barbara L.; Nicholas, Thomas J.; Zhao, Zhongying; Sandel, Matthew J.; Waterston, Robert H.

    2008-01-01

    We describe a system that permits the automated analysis of reporter gene expression in Caenorhabditis elegans with cellular resolution continuously during embryogenesis and demonstrate its utility by defining the expression patterns of reporters for several embryonically expressed transcription factors. The invariant cell lineage permits the automated alignment of multiple expression profiles, allowing the direct comparison of the expression of different genes' reporters. We have also used the system to monitor perturbations to normal development involving changes both in cell division timing and in cell fate. Systematic application could reveal the gene activity of each cell throughout development. PMID:18587405

  20. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Ha, Misook; Hong, Soondo

    2016-04-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

  1. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    PubMed Central

    Ha, Misook; Hong, Soondo

    2016-01-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac. PMID:27075878

  2. Transcriptional Profiles of Imprinted Genes in Human Embryonic Stem Cells During In vitro Differentiation

    PubMed Central

    Park, Sang-Wook; Do, Hyo-Sang; Kim, Dongkyu; Ko, Ji-Yun; Lee, Sang-Hun; Han, Yong-Mahn

    2014-01-01

    Background and Objectives: Genomic imprinting is an inheritance phenomenon by which a subset of genes are expressed from one allele of two homologous chromosomes in a parent of origin-specific manner. Even though fine-tuned regulation of genomic imprinting process is essential for normal development, no other means are available to study genomic imprinting in human during embryonic development. In relation with this bottleneck, differentiation of human embryonic stem cells (hESCs) into specialized lineages may be considered as an alternative to mimic human development. Methods and Results: In this study, hESCs were differentiated into three lineage cell types to analyze temporal and spatial expression of imprinted genes. Of 19 imprinted genes examined, 15 imprinted genes showed similar transcriptional level among two hESC lines and two human induced pluripotent stem cell (hiPSC) lines. Expressional patterns of most imprinted genes were varied in progenitors and fully differentiated cells which were derived from hESCs. Also, no consistence was observed in the expression pattern of imprinted genes within an imprinting domain during in vitro differentiation of hESCs into three lineage cell types. Conclusions: Transcriptional expression of imprinted genes is regulated in a cell type- specific manner in hESCs during in vitro differentiation. PMID:25473448

  3. Two new δ-globin gene variants: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro (HBD: c.44T>C)] and Hb A(2)-Marseille [δ22(B4) Ala→Lys (HBD: c.67G>A;68C>A)].

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Desbrée, Aurélie; Couprie, Nicole; Francina, Alain

    2013-01-01

    We report two new variants of the δ-globin gene: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro] and Hb A(2)-Marseille [δ22(B4)Ala→Lys]. The first variant has a low rate of expression, the second results from a double nucleotide mutation on the same codon. PMID:23227922

  4. Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells

    PubMed Central

    Sritanaudomchai, Hathaitip; Ma, Hong; Clepper, Lisa; Gokhale, Sumita; Bogan, Randy; Hennebold, Jon; Wolf, Don; Mitalipov, Shoukhrat

    2010-01-01

    BACKGROUND Parthenogenetic embryonic stem cells (PESCs) may have future utilities in cell replacement therapies since they are closely related to the female from which the activated oocyte was obtained. Furthermore, the avoidance of parthenogenetic development in mammals provides the most compelling rationale for the evolution of genomic imprinting, and the biological process of parthenogenesis raises complex issues regarding differential gene expression. METHODS AND RESULTS We describe here homozygous rhesus monkey PESCs derived from a spontaneously duplicated, haploid oocyte genome. Since the effect of homozygosity on PESCs pluripotency and differentiation potential is unknown, we assessed the similarities and differences in pluripotency markers and developmental potential by in vitro and in vivo differentiation of homozygous and heterozygous PESCs. To understand the differences in gene expression regulation between parthenogenetic and biparental embryonic stem cells (ESCs), we conducted microarray analysis of genome-wide mRNA profiles of primate PESCs and ESCs derived from fertilized embryos using the Affymetrix Rhesus Macaque Genome array. Several known paternally imprinted genes were in the highly down-regulated group in PESCs compared with ESCs. Furthermore, allele-specific expression analysis of other genes whose expression is also down-regulated in PESCs, led to the identification of one novel imprinted gene, inositol polyphosphate-5-phosphatase F (INPP5F), which was exclusively expressed from a paternal allele. CONCLUSION Our findings suggest that PESCs could be used as a model for studying genomic imprinting, and in the discovery of novel imprinted genes. PMID:20522441

  5. Variability of Gene Expression Identifies Transcriptional Regulators of Early Human Embryonic Development

    PubMed Central

    Hasegawa, Yu; Taylor, Deanne; Ovchinnikov, Dmitry A.; Wolvetang, Ernst J.; de Torrenté, Laurence; Mar, Jessica C.

    2015-01-01

    An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression

  6. Time-Series Interactions of Gene Expression, Vascular Growth and Hemodynamics during Early Embryonic Arterial Development.

    PubMed

    Goktas, Selda; Uslu, Fazil E; Kowalski, William J; Ermek, Erhan; Keller, Bradley B; Pekkan, Kerem

    2016-01-01

    The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD) and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT) imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS) between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis. PMID:27552150

  7. Time-Series Interactions of Gene Expression, Vascular Growth and Hemodynamics during Early Embryonic Arterial Development

    PubMed Central

    Goktas, Selda; Uslu, Fazil E.; Kowalski, William J.; Ermek, Erhan; Keller, Bradley B.

    2016-01-01

    The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD) and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT) imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS) between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis. PMID:27552150

  8. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    SciTech Connect

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  9. Hb St. Jozef, A Val-->Leu N-terminal mutation leading to retention of the methionine, and partial acetylation found in the globin gene in Cis with a -alpha3.7 thalassemia deletion.

    PubMed

    Harteveld, Cornelis L; Versteegh, Florens G A; van Leer, Eduard H G; Starreveld, Jaap S; Kok, Peter J M J; van Rooijen-Nijdam, Irene; van Delft, Peter; Zanella-Cleon, Isabelle; Becchi, Michel; Wajcman, Henri; Giordano, Piero C

    2007-01-01

    We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion. PMID:17654068

  10. Tbx18 targets dermal condensates for labeling, isolation, and gene ablation during embryonic hair follicle formation.

    PubMed

    Grisanti, Laura; Clavel, Carlos; Cai, Xiaoqiang; Rezza, Amelie; Tsai, Su-Yi; Sennett, Rachel; Mumau, Melanie; Cai, Chen-Leng; Rendl, Michael

    2013-02-01

    How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. Although functional analysis of hair follicle epithelial stem cells by gene targeting is well established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking because of the absence of cell type-specific drivers. Here, we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18(LacZ), Tbx18(H2BGFP), and Tbx18(Cre) knock-in mouse models, we demonstrate LacZ and H2BGFP (nuclear green fluorescent protein) expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. As Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we use tamoxifen-inducible Cre-expressing mice, Tbx18(MerCreMer), to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target for the first time embryonic DP precursors for labeling, isolation, and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis. PMID:22992803

  11. Prediction of developmental chemical toxicity based on gene networks of human embryonic stem cells

    PubMed Central

    Yamane, Junko; Aburatani, Sachiyo; Imanishi, Satoshi; Akanuma, Hiromi; Nagano, Reiko; Kato, Tsuyoshi; Sone, Hideko; Ohsako, Seiichiroh; Fujibuchi, Wataru

    2016-01-01

    Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5–100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development. PMID:27207879

  12. Early embryonic failure: Expression and imprinted status of candidate genes on human chromosome 21

    SciTech Connect

    Sherman, L.S.; Bennett, P.R.; Moore, G.E.

    1994-09-01

    Two cases of maternal uniparental (hetero)disomy for human chromosome 21 (mUPD21) have been identified in a systematic search for UPD in 23 cases of early embryonic failure (EEF). Bi-parental origin of the other chromosome pairs was confirmed using specific VNTR probes or dinucleotide repeat analysis. Both maternally and paternally derived isochromosomes 21q have previously been identified in two individuals with normal phenotypes. Full UPD21 has a different mechanism of origin than uniparental isochromosome 21q and its effect on imprinted genes and phenotypic outcome will therefore not necessarily be the same. EEF associated with mUPD21 suggests that developmentally important genes on HSA 21 may be imprinted such that they are only expressed from either the maternally or paternally derived alleles. We have searched for monoallelic expression of candidate genes on HSA 21 in human pregnancy (CBS, IFNAR, COL6A1) using intragenic DNA polymorphisms. These genes were chosen either because their murine homologues lie in imprinted regions or because they are potentially important in embryogenesis. Once imprinted candidate genes have been identified, their methylation status and expression in normal, early embryonic failure and uniparental disomy 21 pregnancies will be studied. At the same time, a larger number of cases of EEF are being examined to further investigate the incidence of UPD21 in this group.

  13. Prediction of developmental chemical toxicity based on gene networks of human embryonic stem cells.

    PubMed

    Yamane, Junko; Aburatani, Sachiyo; Imanishi, Satoshi; Akanuma, Hiromi; Nagano, Reiko; Kato, Tsuyoshi; Sone, Hideko; Ohsako, Seiichiroh; Fujibuchi, Wataru

    2016-07-01

    Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development. PMID:27207879

  14. Ring1b bookmarks genes in pancreatic embryonic progenitors for repression in adult β cells.

    PubMed

    van Arensbergen, Joris; García-Hurtado, Javier; Maestro, Miguel Angel; Correa-Tapia, Miguel; Rutter, Guy A; Vidal, Miguel; Ferrer, Jorge

    2013-01-01

    Polycomb-mediated gene repression is essential for embryonic development, yet its precise role in lineage-specific programming is poorly understood. Here we inactivated Ring1b, encoding a polycomb-repressive complex 1 subunit, in pancreatic multipotent progenitors (Ring1b(progKO)). This caused transcriptional derepression of a subset of direct Ring1b target genes in differentiated pancreatic islet cells. Unexpectedly, Ring1b inactivation in differentiated islet β cells (Ring1b(βKO)) did not cause derepression, even after multiple rounds of cell division, suggesting a role for Ring1b in the establishment but not the maintenance of repression. Consistent with this notion, derepression in Ring1b(progKO) islets occurred preferentially in genes that were targeted de novo by Ring1b during pancreas development. The results support a model in which Ring1b bookmarks its target genes during embryonic development, and these genes are maintained in a repressed state through Ring1b-independent mechanisms in terminally differentiated cells. This work provides novel insights into how epigenetic mechanisms contribute to shaping the transcriptional identity of differentiated lineages. PMID:23271347

  15. An Orthologous Epigenetic Gene Expression Signature Derived from Differentiating Embryonic Stem Cells Identifies Regulators of Cardiogenesis

    PubMed Central

    Busser, Brian W.; Lin, Yongshun; Yang, Yanqin; Zhu, Jun; Chen, Guokai; Michelson, Alan M.

    2015-01-01

    Here we used predictive gene expression signatures within a multi-species framework to identify the genes that underlie cardiac cell fate decisions in differentiating embryonic stem cells. We show that the overlapping orthologous mouse and human genes are the most accurate candidate cardiogenic genes as these genes identified the most conserved developmental pathways that characterize the cardiac lineage. An RNAi-based screen of the candidate genes in Drosophila uncovered numerous novel cardiogenic genes. shRNA knockdown combined with transcriptome profiling of the newly-identified transcription factors zinc finger protein 503 and zinc finger E-box binding homeobox 2 and the well-known cardiac regulatory factor NK2 homeobox 5 revealed that zinc finger E-box binding homeobox 2 activates terminal differentiation genes required for cardiomyocyte structure and function whereas zinc finger protein 503 and NK2 homeobox 5 are required for specification of the cardiac lineage. We further demonstrated that an essential role of NK2 homeobox 5 and zinc finger protein 503 in specification of the cardiac lineage is the repression of gene expression programs characteristic of alternative cell fates. Collectively, these results show that orthologous gene expression signatures can be used to identify conserved cardiogenic pathways. PMID:26485529

  16. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    PubMed Central

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-01

    Summary CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. PMID:26771356

  17. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing

    PubMed Central

    Jacinto, Filipe V.; Benner, Chris; Hetzer, Martin W.

    2015-01-01

    Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing. PMID:26080816

  18. Identification of a stage selector element in the human gamma-globin gene promoter that fosters preferential interaction with the 5' HS2 enhancer when in competition with the beta-promoter.

    PubMed

    Jane, S M; Ney, P A; Vanin, E F; Gumucio, D L; Nienhuis, A W

    1992-08-01

    The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human beta-globin locus control region is required for high level globin gene expression. We investigated interaction between HS2 and the gamma- and beta-promoters using reporter constructs in transient assays in human erythroleukemia (K562) cells. The beta-promoter, usually silent in K562 cells, was activated by HS2. This activity was abolished when a gamma-promoter was linked in cis. Analysis of truncation mutants suggested that sequences conveying the competitive advantage of the gamma-promoter for HS2 included those between positions -53 and -35 relative to the transcriptional start site. This sequence, when used to replace the corresponding region of the beta-promoter, increased beta-promoter activity 10-fold when linked to HS2. The modified beta-promoter was also capable of competing with a gamma-promoter modified internally in the -53 to -35 region, when the two promoters were linked to HS2 in a single plasmid. The corresponding sequences from the Galago gamma-promoter, a species which lacks fetal gamma-gene expression, were inactive in analogous assays. We have identified and partially purified a nuclear protein found in human (fetal stage) erythroleukemia cells, but present in much lower concentration in murine (adult stage) erythroleukemia cells, that binds the -53 to -35 sequence of the gamma-promoter. We speculate that this region of the gamma-promoter functions as a stage selector element in the regulation of hemoglobin switching in humans. PMID:1639067

  19. MicroRNA and gene expression patterns in the differentiation of human embryonic stem cells

    PubMed Central

    Ren, Jiaqiang; Jin, Ping; Wang, Ena; Marincola, Francesco M; Stroncek, David F

    2009-01-01

    Background The unique features of human embryonic stem (hES) cells make them the best candidate resource for both cell replacement therapy and development research. However, the molecular mechanisms responsible for the simultaneous maintenance of their self-renewal properties and undifferentiated state remain unclear. Non-coding microRNAs (miRNA) which regulate mRNA cleavage and inhibit encoded protein translation exhibit temporal or tissue-specific expression patterns and they play an important role in development timing. Results In this study, we analyzed miRNA and gene expression profiles among samples from 3 hES cell lines (H9, I6 and BG01v), differentiated embryoid bodies (EB) derived from H9 cells at different time points, and 5 adult cell types including Human Microvascular Endothelial Cells (HMVEC), Human Umbilical Vein Endothelial Cells (HUVEC), Umbilical Artery Smooth Muscle Cells (UASMC), Normal Human Astrocytes (NHA), and Lung Fibroblasts (LFB). This analysis rendered 104 miRNAs and 776 genes differentially expressed among the three cell types. Selected differentially expressed miRNAs and genes were further validated and confirmed by quantitative real-time-PCR (qRT-PCR). Especially, members of the miR-302 cluster on chromosome 4 and miR-520 cluster on chromosome 19 were highly expressed in undifferentiated hES cells. MiRNAs in these two clusters displayed similar expression levels. The members of these two clusters share a consensus 7-mer seed sequence and their targeted genes had overlapping functions. Among the targeted genes, genes with chromatin structure modification function are enriched suggesting a role in the maintenance of chromatin structure. We also found that the expression level of members of the two clusters, miR-520b and miR-302c, were negatively correlated with their targeted genes based on gene expression analysis Conclusion We identified the expression patterns of miRNAs and gene transcripts in the undifferentiation of human embryonic

  20. Spatio-temporal expression patterns of anterior Hox genes during Nile tilapia (Oreochromis niloticus) embryonic development.

    PubMed

    Lyon, R Stewart; Davis, Adam; Scemama, Jean-Luc

    2013-01-01

    Hox genes encode transcription factors that function to pattern regional tissue identities along the anterior-posterior axis during animal embryonic development. Divergent nested Hox gene expression patterns within the posterior pharyngeal arches may play an important role in patterning morphological variation in the pharyngeal jaw apparatus (PJA) between evolutionarily divergent teleost fishes. Recent gene expression studies have shown the expression patterns from all Hox paralog group (PG) 2-6 genes in the posterior pharyngeal arches (PAs) for the Japanese medaka (Oryzias latipes) and from most genes of these PGs for the Nile tilapia (Oreochromis niloticus). While several orthologous Hox genes exhibit divergent spatial and temporal expression patterns between these two teleost species in the posterior PAs, several tilapia Hox gene expression patterns from PG3-6 must be documented for a full comparative study. Here we present the spatio-temporal expression patterns of hoxb3b, c3a, b4a, a5a, b5a, b5b, b6a and b6b in the neural tube and posterior PAs of the Nile tilapia. We show that several of these tilapia Hox genes exhibit divergent expression patterns in the posterior PAs from their medaka orthologs. We also compare these gene expression patterns to orthologs in other gnathostome vertebrates, including the dogfish shark. PMID:23376031

  1. Characterization of histone H3K27 modifications in the {beta}-globin locus

    SciTech Connect

    Kim, Yea Woon; Kim, AeRi

    2011-02-11

    Research highlights: {yields} The {beta}-globin locus control region is hyperacetylated and monomethylated at histone H3K27. {yields} Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. {yields} Association of PRC2 subunits is comparable with H3K27me3 pattern. {yields} Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human {beta}-globin locus using the ChIP assay. The LCR of the human {beta}-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the {beta}-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  2. Co-inheritance of novel ATRX gene mutation and globin (α & β) gene mutations in transfusion dependent beta-thalassemia patients.

    PubMed

    Al-Nafie, Awatif N; Borgio, J Francis; AbdulAzeez, Sayed; Al-Suliman, Ahmed M; Qaw, Fuad S; Naserullah, Zaki A; Al-Jarrash, Sana; Al-Madan, Mohammed S; Al-Ali, Rudaynah A; AlKhalifah, Mohammed A; Al-Muhanna, Fahad; Steinberg, Martin H; Al-Ali, Amein K

    2015-06-01

    α-Thalassemia X-linked mental retardation syndrome is a rare inherited intellectual disability disorder due to mutations in the ATRX gene. In our previous study of the prevalence of β-thalassemia mutations in the Eastern Province of Saudi Arabia, we confirmed the widespread coinheritance of α-thalassemia mutation. Some of these subjects have a family history of mental retardation, the cause of which is unknown. Therefore, we investigated the presence or absence of mutations in the ATRX gene in these patients. Three exons of the ATRX gene and their flanking regions were directly sequenced. Only four female transfusion dependent β-thalassemia patients were found to be carriers of a novel mutation in the ATRX gene. Two of the ATRX gene mutations, c.623delA and c.848T>C were present in patients homozygous for IVS I-5(G→C) and homozygous for Cd39(C → T) β-thalassemia mutation, respectively. While the other two that were located in the intronic region (flanking regions), were present in patients homozygous for Cd39(C → T) β-thalassemia mutation. The two subjects with the mutations in the coding region had family members with mental retardation, which suggests that the novel frame shift mutation and the missense mutation at coding region of ATRX gene are involved in ATRX syndrome. PMID:25976463

  3. Gene expression patterns in primary neuronal clusters of the Drosophila embryonic brain

    PubMed Central

    Sprecher, Simon G.; Reichert, Heinrich; Hartenstein, Volker

    2014-01-01

    The brain of Drosophila is formed by approximately 100 lineages, each lineage being derived from a stem cell-like neuroblast that segregates from the procephalic neurectoderm of the early embryo. A neuroblast map has been established in great detail for the early embryo, and a suite of molecular markers has been defined for all neuroblasts included in this map (Urbach and Technau, 2003a). However, the expression of these markers was not followed into later embryonic or larval stages, mainly due to the fact that anatomical landmarks to which expression patterns could be related had not been defined. Such markers, in the form of stereotyped clusters of neurons whose axons project along cohesive bundles (“primary axon bundles” or “PABs”) are now available (Younossi-Hartenstein et al., 2006). In the present study we have mapped the expression of molecular markers in relationship to primary neuronal clusters and their PABs. The markers we analyzed include many of the genes involved in patterning of the brain along the anteroposterior axis (cephalic gap genes, segment polarity genes) and dorso-ventral axis (columnar patterning genes), as well as genes expressed in the dorsal protocerebrum and visual system (early eye genes). Our analysis represents an important step along the way to identify neuronal lineages of the mature brain with genes expressed in the early embryo in discrete neuroblasts. Furthermore, the analysis helped us to reconstruct the morphogenetic movements that transform the two-dimensional neuroblast layer of the early embryo into the three-dimensional larval brain and provides the basis for deeper understanding of how the embryonic brain develops. PMID:17300994

  4. Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.

    PubMed

    Mora-Lorca, José Antonio; Sáenz-Narciso, Beatriz; Gaffney, Christopher J; Naranjo-Galindo, Francisco José; Pedrajas, José Rafael; Guerrero-Gómez, David; Dobrzynska, Agnieszka; Askjaer, Peter; Szewczyk, Nathaniel J; Cabello, Juan; Miranda-Vizuete, Antonio

    2016-07-01

    Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. PMID:27117030

  5. Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

    PubMed Central

    Nourizadeh-Lillabadi, Rasoul; Seilø Torgersen, Jacob; Vestrheim, Olav; König, Melanie; Aleström, Peter; Syed, Mohasina

    2010-01-01

    Background The Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. Methodology/Principal Findings The zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO) knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf) as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. Conclusions/Significance The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development. PMID:21042590

  6. Gene Expression Analysis of Mouse Embryonic Stem Cells Following Levitation in an Ultrasound Standing Wave Trap

    PubMed Central

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-01-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08–0.85 MPa) and times (5–60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. (E-mail: Bazoud@tcd.ie) PMID:21208732

  7. Gene expression analysis of mouse embryonic stem cells following levitation in an ultrasound standing wave trap.

    PubMed

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-02-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08-0.85 MPa) and times (5-60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. PMID:21208732

  8. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

    PubMed Central

    Tsuzuki, T; Fujii, Y; Sakumi, K; Tominaga, Y; Nakao, K; Sekiguchi, M; Matsushiro, A; Yoshimura, Y; MoritaT

    1996-01-01

    The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8692798

  9. A new role for muscle segment homeobox genes in mammalian embryonic diapause

    PubMed Central

    Cha, Jeeyeon; Sun, Xiaofei; Bartos, Amanda; Fenelon, Jane; Lefèvre, Pavine; Daikoku, Takiko; Shaw, Geoff; Maxson, Robert; Murphy, Bruce D.; Renfree, Marilyn B.; Dey, Sudhansu K.

    2013-01-01

    Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice—it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness. PMID:23615030

  10. Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid.

    PubMed

    Kashyap, Vasundhra; Gudas, Lorraine J; Brenet, Fabienne; Funk, Patricia; Viale, Agnes; Scandura, Joseph M

    2011-02-01

    Retinoic acid (RA) regulates clustered Hox gene expression during embryogenesis and is required to establish the anterior-posterior body plan. Using mutant embryonic stem cell lines deficient in the RA receptor γ (RARγ) or Hoxa1 3'-RA-responsive element, we studied the kinetics of transcriptional and epigenomic patterning responses to RA. RARγ is essential for RA-induced Hox transcriptional activation, and deletion of its binding site in the Hoxa1 enhancer attenuates transcriptional and epigenomic activation of both Hoxa and Hoxb gene clusters. The kinetics of epigenomic reorganization demonstrate that complete erasure of the polycomb repressive mark H3K27me3 is not necessary to initiate Hox transcription. RARγ is not required to establish the bivalent character of Hox clusters, but RA/RARγ signaling is necessary to erase H3K27me3 from activated Hox genes during embryonic stem cell differentiation. Highly coordinated, long range epigenetic Hox cluster reorganization is closely linked to transcriptional activation and is triggered by RARγ located at the Hoxa1 3'-RA-responsive element. PMID:21087926

  11. A new role for muscle segment homeobox genes in mammalian embryonic diapause.

    PubMed

    Cha, Jeeyeon; Sun, Xiaofei; Bartos, Amanda; Fenelon, Jane; Lefèvre, Pavine; Daikoku, Takiko; Shaw, Geoff; Maxson, Robert; Murphy, Bruce D; Renfree, Marilyn B; Dey, Sudhansu K

    2013-04-01

    Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice-it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness. PMID:23615030

  12. KLF1 stabilizes GATA-1 and TAL1 occupancy in the human β-globin locus.

    PubMed

    Kang, Yujin; Kim, Yea Woon; Yun, Jangmi; Shin, Jongo; Kim, AeRi

    2015-03-01

    KLF1 is an erythroid specific transcription factor that binds to regulatory regions of erythroid genes. Binding sites of KLF1 are often found near binding sites of GATA-1 and TAL1. In the β-globin locus, KLF1 is required for forming active chromatin structure, although its role is unclear. To explore the role of KLF1 in transcribing the human γ-globin genes, we stably reduced the expression of KLF1 in erythroid K562 cells, compromising its association in the β-globin locus. The γ-globin transcription was reduced with disappearance of active chromatin structure of the locus in the KLF1 knockdown cells. Interestingly, GATA-1 and TAL1 binding was reduced in the β-globin locus, even though their expressions were not affected by KLF1 knockdown. The KLF1-dependent GATA-1 and TAL1 binding was observed in the adult locus transcribing the β-globin gene and in several erythroid genes, where GATA-1 occupancy is independent from TAL1. These results indicate that KLF1 plays a role in facilitating and/or stabilizing GATA-1 and TAL1 occupancy in the erythroid genes, contributing to the generation of active chromatin structure such as histone acetylation and chromatin looping. PMID:25528728

  13. The T----C substitution at -198 of the A gamma-globin gene associated with the British form of HPFH generates overlapping recognition sites for two DNA-binding proteins.

    PubMed Central

    Fischer, K D; Nowock, J

    1990-01-01

    Defects in the developmental changes of human hemoglobin production characterized by the continued expression of fetal globin during adult life are classified as hereditary persistence of fetal hemoglobin (HPFH). Among the various molecular lesions associated with this phenotype, the non-deletion forms with point mutations in the promoter region are thought to provide mechanistic clues for gamma-globin gene regulation. The natural occurrence of four different base substitutions mapping within six nucleotides of a homopurine.homopyrimidine motif in the upstream promoter region demarcate a potential control element. To assess its importance for transcriptional activity, we compared the -202 (C----G), -198 (T----C) and -196 (C----T) HPFH mutations with the normal sequence in binding studies with nuclear proteins from erythroid and non-erythroid cells. Wildtype DNA and HPFH mutations at -202 or -196 showed only a weak protein interaction of unclear functional significance. In contrast, -198 (T----C) generated overlapping, high-affinity binding sites for two ubiquitous nuclear proteins. One cognate protein was identified as the transcription factor Sp1. The second one was termed NF-G.C as it interacted strongly with the homopolymer poly(dG).poly(dC). The generation of additional recognition sites for trans-acting factors by the -198 HPFH mutation correlated with a modest increase in promoter activity in vitro specifically with nuclear extracts from erythroid cells. The activation appears to be mediated by binding of Sp1, but it requires interaction with an erythroid-specific factor, most likely GF-1. Templates containing the -196 HPFH mutation showed a transcriptional activity identical to wildtype. This suggests that despite the topological proximity of the mutations, the HPFH phenotype may be established by different mechanisms. Images PMID:1699206

  14. Comparative analysis of temporal gene expression patterns in the developing ovary of the embryonic chicken

    PubMed Central

    YU, Minli; XU, Yali; YU, Defu; YU, Debing; DU, Wenxing

    2015-01-01

    Many genes participate in the process of ovarian germ cell development, while the combined action mechanisms of these molecular regulators still need clarification. The present study was focused on determination of differentially expressed genes and gene functions at four critical time points in chicken ovarian development. Comparative transcriptional profiling of ovaries from embryonic day 5.5 (E5.5), E12.5, E15.5 and E18.5 was performed using an Affymetrix GeneChip chicken genome microarray. Differential expression patterns for genes specifically depleted and enriched in each stage were identified. The results showed that most of the up- and downregulated genes were involved in the metabolism of retinoic acid (RA) and synthesis of hormones. Among them, a higher number of up- and downregulated genes in the E15.5 ovary were identified as being involved in steroid biosynthesis and retinol metabolism, respectively. To validate gene changes, expressions of twelve candidate genes related to germ cell development were examined by real-time PCR and found to be consistent with the of GeneChip data. Moreover, the immunostaining results suggested that ovarian development during different stages was regulated by different genes. Furthermore, a Raldh2 knockdown chicken model was produced to investigate the fundamental role of Raldh2 in meiosis initiation. It was found that meiosis occurred abnormally in Raldh2 knockdown ovaries, but the inhibitory effect on meiosis was reversed by the addition of exogenous RA. This study offers insights into the profile of gene expression and mechanisms regulating ovarian development, especially the notable role of Raldh2 in meiosis initiation in the chicken. PMID:25736178

  15. Systematic identification of gene family regulators in mouse and human embryonic stem cells

    PubMed Central

    Aaronson, Yair; Livyatan, Ilana; Gokhman, David; Meshorer, Eran

    2016-01-01

    Pluripotent self-renewing embryonic stem cells (ESCs) have been the focus of a growing number of high-throughput experiments, revealing the genome-wide locations of hundreds of transcription factors and histone modifications. While most of these datasets were used in a specific context, all datasets combined offer a comprehensive view of chromatin characteristics and regulatory elements that govern cell states. Here, using hundreds of datasets in ESCs, we generated colocalization maps of chromatin proteins and modifications, and built a discovery pipeline for regulatory proteins of gene families. By comparing genome-wide binding data with over-expression and knockdown analysis of hundreds of genes, we discovered that the pluripotency-related factor NR5A2 separates mitochondrial from cytosolic ribosomal genes, regulating their expression. We further show that genes with a common chromatin profile are enriched for distinct Gene Ontology (GO) categories. Our approach can be generalized to reveal common regulators of any gene group; discover novel gene families, and identify common genomic elements based on shared chromatin features. PMID:27084933

  16. Systematic identification of gene family regulators in mouse and human embryonic stem cells.

    PubMed

    Aaronson, Yair; Livyatan, Ilana; Gokhman, David; Meshorer, Eran

    2016-05-19

    Pluripotent self-renewing embryonic stem cells (ESCs) have been the focus of a growing number of high-throughput experiments, revealing the genome-wide locations of hundreds of transcription factors and histone modifications. While most of these datasets were used in a specific context, all datasets combined offer a comprehensive view of chromatin characteristics and regulatory elements that govern cell states. Here, using hundreds of datasets in ESCs, we generated colocalization maps of chromatin proteins and modifications, and built a discovery pipeline for regulatory proteins of gene families. By comparing genome-wide binding data with over-expression and knockdown analysis of hundreds of genes, we discovered that the pluripotency-related factor NR5A2 separates mitochondrial from cytosolic ribosomal genes, regulating their expression. We further show that genes with a common chromatin profile are enriched for distinct Gene Ontology (GO) categories. Our approach can be generalized to reveal common regulators of any gene group; discover novel gene families, and identify common genomic elements based on shared chromatin features. PMID:27084933

  17. MicroRNA-10 modulates Hox genes expression during Nile tilapia embryonic development.

    PubMed

    Giusti, Juliana; Pinhal, Danillo; Moxon, Simon; Campos, Camila Lovaglio; Münsterberg, Andrea; Martins, Cesar

    2016-05-01

    Hox gene clusters encode a family of transcription factors that govern anterior-posterior axis patterning during embryogenesis in all bilaterian animals. The time and place of Hox gene expression are largely determined by the relative position of each gene within its cluster. Furthermore, Hox genes were shown to have their expression fine-tuned by regulatory microRNAs (miRNAs). However, the mechanisms of miRNA-mediated regulation of these transcription factors during fish early development remain largely unknown. Here we have profiled three highly expressed miR-10 family members of Nile tilapia at early embryonic development, determined their genomic organization as well as performed functional experiments for validation of target genes. Quantitative analysis during developmental stages showed miR-10 family expression negatively correlates with the expression of HoxA3a, HoxB3a and HoxD10a genes, as expected for bona fide miRNA-mRNA interactions. Moreover, luciferase assays demonstrated that HoxB3a and HoxD10a are targeted by miR-10b-5p. Overall, our data indicate that the miR-10 family directly regulates members of the Hox gene family during Nile tilapia embryogenesis. PMID:26980108

  18. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    PubMed Central

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior. PMID:27148349

  19. Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

    PubMed Central

    Tsakiridis, Anestis; Tzouanacou, Elena; Rahman, Afifah; Colby, Douglas; Axton, Richard; Chambers, Ian; Wilson, Valerie; Forrester, Lesley; Brickman, Joshua M.

    2009-01-01

    Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting. PMID:19692586

  20. Functional Genomics Screening Utilizing Mutant Mouse Embryonic Stem Cells Identifies Novel Radiation-Response Genes

    PubMed Central

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C.; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy. PMID:25853515

  1. alpha-Thalassemia caused by an unstable alpha-globin mutant.

    PubMed Central

    Liebhaber, S A; Kan, Y W

    1983-01-01

    In a previous study, molecular cloning of the alpha-globin genes from a patient with nondeletion Hb-H disease (genotype--/alpha alpha) showed that a single nucleotide mutation (CTG to CCG) in one of the genes resulted in a leucine to proline substitution. This paper describes the approach we used to detect the abnormal alpha-globin chain. The chain was identified using a cell-free translation system. It turned over rapidly both in vitro and in vivo in the patient's reticulocytes. The unusual feature of this unstable alpha-globin is that the alpha-globin deficiency causes alpha-thalassemia. Simple heterozygotes for this lesion (alpha Pro alpha/alpha alpha) resemble alpha-thalassemia carriers and do not exhibit the hemolytic anemia usually associated with unstable hemoglobins. Images PMID:6826718

  2. Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms.

    PubMed

    Dai, Yan; Sangerman, Jose; Luo, Hong Yuan; Fucharoen, Suthat; Chui, David H K; Faller, Douglas V; Perrine, Susan P

    2016-01-01

    Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy. PMID:26603726

  3. Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation.

    PubMed

    Mabaera, Rodwell; Richardson, Christine A; Johnson, Kristin; Hsu, Mei; Fiering, Steven; Lowrey, Christopher H

    2007-08-15

    The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching. PMID:17456718

  4. Gene expression profiles during early differentiation of mouse embryonic stem cells

    PubMed Central

    Mansergh, Fiona C; Daly, Carl S; Hurley, Anna L; Wride, Michael A; Hunter, Susan M; Evans, Martin J

    2009-01-01

    Background Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1–4 EBs Results An initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile. Conclusion ES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these

  5. Gene expression of Hsps in normal and abnormal embryonic development of mouse hindlimbs.

    PubMed

    Yan, Zhengli; Wei, Huimiao; Ren, Chuanlu; Yuan, Shishan; Fu, Hu; Lv, Yuan; Zhu, Yongfei; Zhang, Tianbao

    2015-06-01

    Heat shock proteins (Hsps), which have important biological functions, are a class of highly conserved genetic molecules with the capacity of protecting and promoting cells to repair themselves from damage caused by various stimuli. Our previous studies found that Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10, and Hsp40 may be related to all-trans retinoic acid (atRA)-induced phocomelic and other abnormalities, while HspA12B, HspA14, Trap1, and Hsp105 may be forelimb development-related genes; Grp78 may play an important role in forelimb development. In this study, the embryonic phocomelic, oligodactylic model of both forelimbs and hindlimbs was developed by atRA administered per os to the pregnant mice on gestational day 11, and the expression of 36 members of Hsps family in normal and abnormal development of embryonic hindlimbs was measured by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). It is found that HspA1L, Hsp22, Hsp10, Hsp60, Hsp47, HspB2, HspB10, HspA12A, Apg1, HspB4, Grp78, and HspB9 probably performs a major function in limb development, and HspA13, Grp94 and Hsp110 may be hindlimb development-related genes. PMID:25352652

  6. Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene

    PubMed Central

    Czerwinska, Areta M.; Grabowska, Iwona; Archacka, Karolina; Bem, Joanna; Swierczek, Barbara; Helinska, Anita; Streminska, Wladyslawa; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Koblowska, Marta

    2016-01-01

    The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs. PMID:26649785

  7. Characterization of tweety gene (ttyh1-3) expression in Xenopus laevis during embryonic development

    PubMed Central

    Rabe, Brian A.; Huyck, Ryan W.; Williams, Cheyenne C.; Saha, Margaret S.

    2015-01-01

    The tweety family of genes encodes large-conductance chloride channels and has been implicated in a wide array of cellular processes including cell division, cell adhesion, regulation of calcium activity, and tumorigenesis, particularly in neuronal cells. However, their expression patterns during early development remain largely unknown. Here, we describe the spatial and temporal patterning of ttyh1, ttyh2, and ttyh3 in Xenopus laevis during early embryonic development. Ttyh1 and ttyh3 are initially expressed at the late neurula stage are and primarily localized to the developing nervous system; however ttyh1 and ttyh3 both show transient expression in the somites. By swimming tadpole stages, all three genes are expressed in the brain, spinal cord, eye, and cranial ganglia. While ttyh1 is restricted to proliferative, ventricular zones, ttyh3 is primarily localized to postmitotic regions of the developing nervous system. Ttyh2, however, is strongly expressed in cranial ganglia V, VII, IX and X. The differing temporal and spatial expression patterns of ttyh1, ttyh2, and ttyh3 suggest that they may play distinct roles throughout embryonic development. PMID:25541457

  8. MIWI2 as an Effector of DNA Methylation and Gene Silencing in Embryonic Male Germ Cells.

    PubMed

    Kojima-Kita, Kanako; Kuramochi-Miyagawa, Satomi; Nagamori, Ippei; Ogonuki, Narumi; Ogura, Atsuo; Hasuwa, Hidetoshi; Akazawa, Takashi; Inoue, Norimitsu; Nakano, Toru

    2016-09-13

    During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon. PMID:27626653

  9. A Gene Trap Knockout of the Tiam-1 Protein Results in Malformation of the Early Embryonic Brain

    PubMed Central

    Yoo, Sooyeon; Kim, Yujin; Lee, Haeryung; Park, Sungjeong; Park, Soochul

    2012-01-01

    Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the β-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice. PMID:22661025

  10. Comprehensive Gene Expression Analysis of Human Embryonic Stem Cells during Differentiation into Neural Cells

    PubMed Central

    Fathi, Ali; Hatami, Maryam; Hajihosseini, Vahid; Fattahi, Faranak; Kiani, Sahar; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2011-01-01

    Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFβ family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation. PMID:21829537

  11. Differential and stage-related expression in embryonic tissues of a new human homoeobox gene.

    PubMed

    Mavilio, F; Simeone, A; Giampaolo, A; Faiella, A; Zappavigna, V; Acampora, D; Poiana, G; Russo, G; Peschle, C; Boncinelli, E

    The homoeobox is a 183 base-pair (bp) DNA sequence conserved in several Drosophila genes controlling segmentation and segment identity. Homoeobox sequences have been detected in the genome of species ranging from insects and anellids to vertebrates and homoeobox containing genes have been cloned from Xenopus, mouse and man. We recently isolated human homoeobox containing complementary DNA clones, that represent transcripts from four different human genes. One clone (HHO.c10) is selectively expressed in a 2.1 kilobase (kb) polyadenylated transcript in the spinal cord of human embryos and fetuses 5-10 weeks after fertilization. We report the characterization of a second cDNA clone, termed HHO.c13, that represents a new homoeobox gene. This clone encodes a protein of 255 amino-acid residues, which includes a pentapeptide, upstream of the homoeo domain, conserved in other Drosophila, Xenopus, murine and human homoeobox genes. By Northern analysis HHO.c13 detects multiple embryonic transcripts, which are differentially expressed in spinal cord, brain, backbone rudiments, limb buds and heart in 5-9-week-old human embryos and fetuses, in a striking organ- and stage-specific pattern. These observations suggest that in early mammalian development homoeobox genes may exert a wide spectrum of control functions in a variety of organs and body parts, in addition to the spinal cord. PMID:2879245

  12. Dissecting the heterogeneity of gene expressions in mouse embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Zou, Ling-Nan; Thomson, Matt; Liu, S. John; Ramanathan, Sharad

    2011-03-01

    A population of genetically identical cells, of the same nominal cell type, and cultured in the same petri dish, will nevertheless often exhibit varying patterns of gene expression. Taking mouse embryonic stem (ES) cells as a model system, we use immunofluorescence and flow cytometry to examine in detail the distribution of expression levels for various transcription factors key to the maintenance of the ES cell identity. We find the population-level distribution of many proteins, once rescaled by the average expression level, have very similar shapes. This suggest the largest component of observed heterogeneity comes from a single source. More subtly, we find the expression many of genes appears to modulate with the cell cycle. This may suggest that the program for maintaining ES cell identity is tightly coupled to the cell cycle machinery. This work is supported by the Harvard Stem Cell Institute and the Jane Coffin Childs Memorial Fund for Medical Research.

  13. Gene-Environment Interactions, Folate Metabolism and the Embryonic Nervous System

    PubMed Central

    Ross, M. Elizabeth

    2010-01-01

    Formation of brain and spinal cord requires the successful closure of neural ectoderm into an embryonic neural tube. Defects in this process result in anencephaly or spina bifida, which together constitute a leading cause of mortality and morbidity in children, affecting all ethnic and socioeconomic groups. The subject of intensive research for decades, neural tube defects (NTDs) are understood to arise from complex interactions of genes and environmental conditions, though systems-level details are still elusive. Despite the variety of underlying causes, a single intervention, folic acid supplementation given in the first gestational month can measurably reduce the occurrence of NTDs in a population. Evidence for and the scope of gene-environment interactions in the genesis of NTDs are discussed. A systems-based approach is now possible toward studies of genetic and environmental influences underlying NTDs that will enable the assessment of individual risk and personalized optimization of prevention. PMID:20836042

  14. Identification and Characterization of Genes Involved in Embryonic Crystal Cell Formation During Drosophila Hematopoiesis

    PubMed Central

    Milchanowski, Allison B.; Henkenius, Amy L.; Narayanan, Maya; Hartenstein, Volker; Banerjee, Utpal

    2004-01-01

    Parallels between vertebrate and Drosophila hematopoiesis add to the value of flies as a model organism to gain insights into blood development. The Drosophila hematopoietic system is composed of at least three classes of terminally differentiated blood cells: plasmatocytes, crystal cells, and lamellocytes. Recent studies have identified transcriptional and signaling pathways in Drosophila involving proteins similar to those seen in human blood development. To identify additional genes involved in Drosophila hematopoiesis, we have conducted a P-element-based genetic screen to isolate mutations that affect embryonic crystal cell development. Using a marker of terminally differentiated crystal cells, we screened 1040 P-element-lethal lines located on the second and third chromosomes and identified 44 individual lines that affect crystal cell development. Identifying novel genes and pathways involved in Drosophila hematopoiesis is likely to provide further insights into mammalian hematopoietic development and disorders. PMID:15454546

  15. Divergent RNA Localisation Patterns of Maternal Genes Regulating Embryonic Patterning in the Butterfly Pararge aegeria

    PubMed Central

    Carter, Jean-Michel; Gibbs, Melanie; Breuker, Casper J.

    2015-01-01

    The maternal effect genes responsible for patterning the embryo along the antero-posterior (AP) axis are broadly conserved in insects. The precise function of these maternal effect genes is the result of the localisation of their mRNA in the oocyte. The main developmental mechanisms involved have been elucidated in Drosophila melanogaster, but recent studies have shown that other insect orders often diverge in RNA localisation patterns. A recent study has shown that in the butterfly Pararge aegeria the distinction between blastodermal embryonic (i.e. germ band) and extra-embryonic tissue (i.e. serosa) is already specified in the oocyte during oogenesis in the ovariole, long before blastoderm cellularisation. To examine the extent by which a female butterfly specifies and patterns the AP axis within the region fated to be the germ band, and whether she specifies a germ plasm, we performed in situ hybridisation experiments on oocytes in P. aegeria ovarioles and on early embryos. RNA localisation of the following key maternal effect genes were investigated: caudal (cad), orthodenticle (otd), hunchback (hb) and four nanos (nos) paralogs, as well as TDRD7 a gene containing a key functional domain (OST-HTH/LOTUS) shared with oskar. TDRD7 was mainly confined to the follicle cells, whilst hb was exclusively zygotically transcribed. RNA of some of the nos paralogs, otd and cad revealed complex localisation patterns within the cortical region prefiguring the germ band (i.e. germ cortex). Rather interestingly, otd was localised within and outside the anterior of the germ cortex. Transcripts of nos-O formed a distinct granular ring in the middle of the germ cortex possibly prefiguring the region where germline stem cells form. These butterfly RNA localisation patterns are highly divergent with respect to other insects, highlighting the diverse ways in which different insect orders maternally regulate early embryogenesis of their offspring. PMID:26633019

  16. Signed weighted gene co-expression network analysis of transcriptional regulation in murine embryonic stem cells

    PubMed Central

    Mason, Mike J; Fan, Guoping; Plath, Kathrin; Zhou, Qing; Horvath, Steve

    2009-01-01

    Background Recent work has revealed that a core group of transcription factors (TFs) regulates the key characteristics of embryonic stem (ES) cells: pluripotency and self-renewal. Current efforts focus on identifying genes that play important roles in maintaining pluripotency and self-renewal in ES cells and aim to understand the interactions among these genes. To that end, we investigated the use of unsigned and signed network analysis to identify pluripotency and differentiation related genes. Results We show that signed networks provide a better systems level understanding of the regulatory mechanisms of ES cells than unsigned networks, using two independent murine ES cell expression data sets. Specifically, using signed weighted gene co-expression network analysis (WGCNA), we found a pluripotency module and a differentiation module, which are not identified in unsigned networks. We confirmed the importance of these modules by incorporating genome-wide TF binding data for key ES cell regulators. Interestingly, we find that the pluripotency module is enriched with genes related to DNA damage repair and mitochondrial function in addition to transcriptional regulation. Using a connectivity measure of module membership, we not only identify known regulators of ES cells but also show that Mrpl15, Msh6, Nrf1, Nup133, Ppif, Rbpj, Sh3gl2, and Zfp39, among other genes, have important roles in maintaining ES cell pluripotency and self-renewal. We also report highly significant relationships between module membership and epigenetic modifications (histone modifications and promoter CpG methylation status), which are known to play a role in controlling gene expression during ES cell self-renewal and differentiation. Conclusion Our systems biologic re-analysis of gene expression, transcription factor binding, epigenetic and gene ontology data provides a novel integrative view of ES cell biology. PMID:19619308

  17. Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression

    PubMed Central

    Lee, Tze Yan; Muniandy, Logeswaran; Teh, Lai Kuan; Abdullah, Maha; George, Elizabeth; Sathar, Jameela; Lai, Mei I

    2016-01-01

    Objective: The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia. PMID:26377036

  18. [γ-Globin Inductive Therapy of β-thalassemia and Its Relationship with MicroRNA].

    PubMed

    Li, Yao-Yao; Gu, Jian; Yu, Duo-Nan

    2016-04-01

    β-thalassemia is a chronic hemolytic anemia characterized by the reduction or absence of synthesis of β-globin chains because of the β-globin gene mutations. β-thalassemia belongs to the inherited hemoglobin disease, and occurs in some provinces of China, such as in Guangdong, Guangxi, Fujian, its prevalence is about 2%. The treatment of this disease include transfusion, iron chelating agent, hematopoietic stem cell transplantation, splenectomy, induced expression of Fetal Hemoglobin (HbF) and gene therapies. However, the mortality rate of this disease is still higher, thus some new treatments are urgently needed. In recent years, the study was mainly concentrated in 2 aspects: the normal β-globin gene transfer and endogenous γ-globin re-activation. Some studies showed that the expression of miRNAs was dysregulated in β-thalassemia. Some miRNAs could regulate γ-globin at posttranscriptional level, thus, the clarification of relationship between miRNAs and β-thalassemia is expected to provide experimental bases to β-thalassemia therapy. In this review, the induced therapy of γ-globin for β-thalassemia and its relationship with the miRNA are summarized. PMID:27151042

  19. Different gene transfer methods at the very early, early, late and whole embryonic stages in chicken.

    PubMed

    Gong, Ping; Yang, Y P; Yang, Y; Feng, Yan P; Li, S J; Peng, Xiu L; Gong, Y Z

    2012-12-01

    New technologies in gene transfer combined with experimental embryology make the chicken embryo an excellent model system for gene function studies. The techniques of in ovo electroporation, in vitro culture for ex ovo electroporation and retrovirus-mediated gene transfer have already been fully developed in chicken. Yet to our knowledge, there are no definite descriptions on the features and application scopes of these techniques. The survival rates of different in vitro culture methods were compared and the EGFP expression areas of different gene transfer techniques were explored. It was that the optimal timings of removing embryo for EC culture and Petri dish system was at E1.5 and E2.5, respectively; and optimal timing of injecting retrovirus is at E0. Results indicated that the EC culture, in ovo electroporation, the Petri dish system and retrovirus-mediated method are, respectively, suitable for the very early, early, late and whole embryonic stages in chicken. Comparison of different gene transfer methods and establishment of optimal timings are expected to provide a better choice of the efficient method for a particular experiment. PMID:23134602

  20. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors

    PubMed Central

    Suzuki, Keiichiro; Mitsui, Kaoru; Aizawa, Emi; Hasegawa, Kouichi; Kawase, Eihachiro; Yamagishi, Toshiyuki; Shimizu, Yoshihiko; Suemori, Hirofumi; Nakatsuji, Norio; Mitani, Kohnosuke

    2008-01-01

    Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was ≈100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only ≈1% with other gene delivery methods. Furthermore, in combination with negative selection, ≈45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells. PMID:18768795

  1. Chibby functions in Xenopus ciliary assembly, embryonic development, and the regulation of gene expression

    PubMed Central

    Shi, Jianli; Zhao, Ying; Galati, Domenico; Winey, Mark; Klymkowsky, Michael W.

    2015-01-01

    Wnt signaling and ciliogenesis are core features of embryonic development in a range of metazoans. Chibby (Cby), a basal-body associated protein, regulates β-catenin-mediated Wnt signaling in the mouse but not Drosophila. Here we present an analysis of Cby’s embryonic expression and morphant phenotypes in Xenopus laevis. Cby RNA is supplied maternally, negatively regulated by Snail2 but not Twist1, preferentially expressed in the neuroectoderm, and regulates β-catenin-mediated gene expression. Reducing Cby levels reduced the density of multiciliated cells, the number of basal bodies per multiciliated cell, and the numbers of neural tube primary cilia; it also led to abnormal development of the neural crest, central nervous system, and pronephros, all defects that were rescued by a Cby-GFP chimera. Reduction of Cby led to an increase in Wnt8a and decreases in Gli2, Gli3, and Shh RNA levels. Many, but not all, morphant phenotypes were significantly reversed by the Wnt inhibitor SFRP2. These observations extend our understanding of Cby’s role in mediating the network of interactions between ciliogenesis, signaling systems and tissue patterning. PMID:25220153

  2. Haploinsufficiency screen highlights two distinct groups of ribosomal protein genes essential for embryonic stem cell fate

    PubMed Central

    Fortier, Simon; MacRae, Tara; Bilodeau, Mélanie; Sargeant, Tobias; Sauvageau, Guy

    2015-01-01

    In a functional genomics screen of mouse embryonic stem cells (ESCs) with nested hemizygous chromosomal deletions, we reveal that ribosomal protein (RP) genes are the most significant haploinsufficient determinants for embryoid body (EB) formation. Hemizygocity for three RP genes (Rps5, Rps14, or Rps28), distinguished by the proximity of their corresponding protein to the ribosome's mRNA exit site, is associated with the most profound phenotype. This EB phenotype was fully rescued by BAC or cDNA complementation but not by the reduction of p53 levels, although such reduction was effective with most other RP-deleted clones corresponding to non-mRNA exit-site proteins. RNA-sequencing studies further revealed that undifferentiated ESCs hemizygous for Rps5 showed reduced expression levels of several mesoderm-specific genes as compared with wild-type counterparts. Together, these results reveal that RP gene dosage limits the differentiation, not the self-renewal, of mouse ESCs. They also highlight two separate mechanisms underlying this process, one of which is p53 independent. PMID:25646475

  3. Evaluation of neural gene expression in serum treated embryonic stem cells in Alzheimer's patients

    PubMed Central

    Dehghani, Leila; Hashemi-Beni, Batool; Poorazizi, Elahe; Khorvash, Fariborz; Shaygannejad, Vahid; Sedghi, Maryam; Vesal, Sahar; Meamar, Rokhsareh

    2013-01-01

    Background: Previous studies confirmed that neural gene expression in embryonic stem cells (ESC) could influence by chemical compounds through stimulating apoptotic pathway. We aimed to use ESCs-derived neural cells by embryoid body formation as an in vitro model for determination of neural gene expression changes in groups that treated by sera from Alzheimer's patients and compare with healthy individuals. Materials and Methods: ESC line which was derived from the C57BL/6 mouse strain was used throughout this study. ESC-derived neural cells were treated with serum from Alzheimer's patient and healthy individual. Neural gene expression was assessed in both groups by quantitative real-time polymerase chain reaction analysis. The data was analyzed by SPSS Software (version 18). Results: Morphologically, the reducing in neurite out-growth was observed in neural cells in group, which treated by serum from Alzheimer's patient, while neurite growth was natural in appearance in control group. Microtubule-associated protein 2 and glial fibrillary acidic protein expression significantly reduced in the Alzheimer's patient group compared with the control group. Nestin expression did not significantly differ among the groups. Conclusion: Neural gene expression could be reduced in serum treated ESC in Alzheimer's patients. PMID:23961278

  4. Expression and regulation of Groucho-related genes in the embryonic chicken feather bud.

    PubMed

    Houghton, Leslie; Freeman, Allison; Morgan, Bruce A

    2003-04-01

    The groucho-related gene (Grg) products modulate the transcriptional response to several extracellular signals, including the Wnts. In an effort to define the roles of Grgs in the morphogenesis of the feather bud, cDNAs encoding members of the Grg family were cloned from embryonic chick skin. In situ hybridization was used to localize transcripts for cGrg2, Grg3, Grg4, and Grg5 in embryos from day 6 through day 9. Expression of cGrg2, 3, and 5 is detected throughout the initial epidermal placode. As the buds mature, expression becomes limited to the posterior halves and eventually to the distal tip of the outgrowing bud. This pattern and the effects of forced activation of the bone morphogenetic protein and beta-catenin signal transduction pathways on Grg gene expression suggest that these genes act downstream of the early activation of the beta-catenin pathway that initiates placode formation. Induction of Grg genes by beta-catenin may serve as a negative feedback to modulate pathway activation while also altering the activity of other transduction pathways involved in bud patterning. PMID:12666196

  5. Embryonic stem cell gene expression signatures in the canine mammary tumor: a bioinformatics approach.

    PubMed

    Zamani-Ahmadmahmudi, Mohamad

    2016-08-01

    Canine breast cancer was considered as an ideal model of comparative oncology for the human breast cancer, as there is significant overlap between biological and clinical characteristics of the human and canine breast cancer. We attempt to clarify expression profile of the embryonic stem cell (ES) gene signatures in canine breast cancer. Using microarray datasets (GSE22516 and GSE20718), expression of the three major ES gene signatures (modules or gene-sets), including Myc, ESC-like, and PRC modules, was primarily analyzed through Gene-Set Enrichment Analysis (GSEA) method in tumor and healthy datasets. For confirmation of the primary results, an additional 13 ES gene-sets which were categorized into four groups including ES expressed (ES exp1 and ES exp2), NOS targets (Nanog targets, Oct4 targets, Sox2 targets, NOS targets, and NOS TFs), Polycomb targets (Suz12 targets, Eed targets, H3K27 bound, and PRC2 targets), and Myc targets (Myc targets1, and Myc targets2) were tested in the tumor and healthy datasets. Our results revealed that there is a valuable overlap between canine and human breast cancer ES gene-sets expression profile, where Myc and ESC-like modules were up-regulated and PRC module was down-regulated in metastatic canine mammary gland tumors. Further analysis of the secondary gene-sets indicated overexpression of the ES expressed, NOS targets (Nanog targets, Oct4 targets, Sox2 targets, and NOS targets), and Myc targets and underexpression of the Polycomb targets in metastatic canine breast cancer. PMID:27307036

  6. RNA Genes: Retroelements and Virally Retroposable microRNAs in Human Embryonic Stem Cells

    PubMed Central

    Fujii, Yoichi R.

    2010-01-01

    Embryonic stem cells (ESCs) are capable of undergoing self-renewal, and their developmental ability is known as the stemness. Recently, microRNAs (miRNAs) as regulators have been isolated from ESCs. Although Dicer and DiGeorge syndrome critical region gene 8 (DGCR8) are essential factors for the biogeneration of miRNA, Dicer-knockout (KO) ESCs have showed to fail to express differentiation markers and DGCR8-KO ESCs have showed to be arrest in the G1 phase. Furthermore, Dicer-KO ESCs lost the ability to epigenetically silence retroelemtns (REs). REs are expressed and transposed in ESCs, whose transcripts control expression of miRNAs, and their transposable retroelement (TE) expression is, therefore related to ESC proliferation and differentiation, suggesting that the interplay between miRNAs and REs may have a deep responsibility for the stemness including a short G1/S transition and for RE regulation in ESCs. PMID:20835360

  7. Specification of select hypothalamic circuits and innate behaviors by the embryonic patterning gene Dbx1

    PubMed Central

    Sokolowski, Katie; Esumi, Shigeyuki; Hirata, Tsutomu; Kamal, Yasman; Tran, Tuyen; Lam, Andrew; Oboti, Livio; Brighthaupt, Sherri-Chanelle; Zaghlula, Manar; Martinez, Jennifer; Ghimbovschi, Svetlana; Knoblach, Susan; Pierani, Alessandra; Tamamaki, Nobuaki; Shah, Nirao M; Jones, Kevin S; Corbin, Joshua G

    2015-01-01

    SUMMARY The hypothalamus integrates information required for the production of a variety of innate behaviors such as feeding, mating, aggression and predator avoidance. Despite an extensive knowledge of hypothalamic function, how embryonic genetic programs specify circuits that regulate these behaviors remains unknown. Here, we find that in the hypothalamus the developmentally regulated homeodomain-containing transcription factor Dbx1 is required for the generation of specific subclasses of neurons within the lateral hypothalamic area/zona incerta (LH) and the arcuate (Arc) nucleus. Consistent with this specific developmental role, Dbx1 hypothalamic-specific conditional-knockout mice display attenuated responses to predator odor and feeding stressors but do not display deficits in other innate behaviors such as mating or conspecific aggression. Thus, activity of a single developmentally regulated gene, Dbx1, is a shared requirement for the specification of hypothalamic nuclei governing a subset of innate behaviors. PMID:25864637

  8. Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures

    SciTech Connect

    Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Fonteyne, Liset J.J. de la; Brauers, Karen J.J.; Claessen, Sandra; Delft, Joost H. van; Kleinjans, Jos C.S.; Piersma, Aldert H.

    2011-03-01

    The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 {mu}M) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

  9. Muscle segment homeobox genes direct embryonic diapause by limiting inflammation in the uterus

    SciTech Connect

    Cha, Jeeyeon; Burnum-Johnson, Kristin E.; Bartos, Amanda; Li, Yingju; Baker, Erin Shammel; Tilton, Susan C.; Webb-Robertson, Bobbie-Jo M.; Piehowski, Paul D.; Monroe, Matthew E.; Jegga, Anil; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K.

    2015-06-11

    Embryonic diapause (delayed implantation) is a reproductive strategy widespread in the animal kingdom. Under this condition, embryos at the blastocyst stage become dormant simultaneously with uterine quiescence until environmental or physiological conditions are favorable for the survival of the mother and newborn. Under favorable conditions, activation of the blastocyst and uterus ensues with implantation and progression of pregnancy. Although endocrine factors are known to participate in this process, the underlying molecular mechanism coordinating this phenomenon is not clearly understood. We recently found that uterine muscle segment homeobox (Msx) transcription factors are critical for the initiation and maintenance of delayed implantation in mice. To better understand why Msx genes are critical for delayed implantation, we compared uterine proteomics profiles between littermate floxed (Msx1/Msx2f/f) mice and mice with uterine deletion of Msx genes (Msx1/Msx2d/d) under delayed conditions. In Msx1/Msx2d/d uteri, pathways including protein translation, ubiquitin-proteasome system, inflammation, chaperone-mediated protein folding, and endoplasmic reticulum (ER) stress were enriched, and computational modeling showed intersection of these pathways on inflammatory responses. Indeed, increases in the ubiquitin-proteasome system and inflammation conformed to proteotoxic and ER stress in Msx1/Msx2d/d uteri under delayed conditions. Interestingly, treatment with a proteasome inhibitor bortezomib further exacerbated ER stress in Msx1/Msx2d/d uteri with aggravated inflammatory response, deteriorating rate of blastocyst recovery and failure to sustain delayed implantation. This study highlights a previously unrecognized role for Msx in preventing proteotoxic stress and inflammatory responses to coordinate embryo dormancy and uterine quiescence during embryonic diapause.

  10. Gene-modified embryonic stem cell test to characterize chemical risks.

    PubMed

    Kitada, Kohei; Kizu, Akane; Teramura, Takeshi; Takehara, Toshiyuki; Hayashi, Masami; Tachibana, Daisuke; Wanibuchi, Hideki; Fukushima, Shoji; Koyama, Masayasu; Yoshida, Kayo; Morita, Takashi

    2015-11-01

    A high-throughput test of cell growth inhibition was performed using mouse embryonic stem (ES) cells to assess chemical toxicities. We herein demonstrated using a 96-well culture plate approach and the MTT assay that this method was suitable for prioritization of chemicals for their cytotoxic properties. In order to categorize chemicals, we used p53 gene-modified mouse ES cells as well as wild-type ES cells. The p53 gene is a well-known tumor suppressor and controls programmed cell death (apoptosis) and cellular senescence that is triggered by DNA-damaging agents such as alkylating agents and radiation. In the present study, p53-deficient ES cells were found to be more resistant to a tumor initiator, diethylnitrosamine (DEN), than wild-type ES cells, suggesting the inhibition of apoptosis or senescence by a dysfunction in p53. Chromosome aberrations were more frequently detected in p53-deficient ES cells than in wild-type cells, indicating genomic instability due to the deletion of p53. Other tumor initiators, methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (NMU), did not reveal apparent differences in cytotoxicity between wild-type and p53-deficient ES cells. Thus, ES test system using gene-modified ES cells may be used to categorize chemicals by detecting their characteristic effects on apoptosis, genotoxic potentials as well as general cytotoxicity. PMID:26206128

  11. Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

    SciTech Connect

    Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith , E-Mail: Judith.hall@ncl.ac.uk

    2005-09-16

    In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.

  12. Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation.

    PubMed

    Simandi, Zoltan; Horvath, Attila; Nagy, Peter; Nagy, Laszlo

    2016-01-01

    Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system. PMID:27403939

  13. Structural organization and chromosomal assignment of the mouse embryonic TEA domain-containing factor (ETF) gene

    SciTech Connect

    Suzuki, Kazuo; Yasunami, Michio; Matsuda, Yoichi

    1996-09-01

    Embryonic TEA domain-containing factor (ETF) belongs to the family of proteins structurally related to transcriptional enhancer factor-1 (TEF-1) and is implicated in neural development. Isolation and characterization of the cosmid clones encoding the mouse ETF gene (Etdf) revealed that Etdf spans approximately 17.9 kb and consists of 12 exons. The exon-intron structure of Etdf closely resembles that of the Drosophila scalloped gene, indicating that these genes may have evolved from a common ancestor. Then multiple transcription initiation sites revealed by S1 protection and primer extension analyses are consistent with the absence of the canonical TATA and CAAT boxes in the 5{prime}-flanking region, which contains many potential regulatory sequences, such as the E-box, N-box, Sp1 element, GATA-1 element, TAATGARAT element, and B2 short interspersed element (SINE) as well as several direct and inverted repeat sequences. The Etdf locus was assigned to the proximal region of mouse chromosome 7 using fluorescence in situ hybridization and linkage mapping analyses. These results provide the molecular basis for studying the regulation, in vivo function, and evolution of Etdf. 29 refs., 5 figs., 1 tab.

  14. The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Liu, Zhong; Zhao, Rui; Giles, Keith E.

    2016-01-01

    It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ~50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor, UBTF, from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency, hESCs were treated with the Pol I inhibitor, CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers, reduces the expression of pluripotency markers, and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene, and corresponding reduction in transcription, represent early regulatory events during the directed differentiation of pluripotent stem cells. PMID:27299313

  15. Cationic, helical polypeptide-based gene delivery for IMR-90 fibroblasts and human embryonic stem cells

    PubMed Central

    Yen, Jonathan; Zhang, Yanfeng; Gabrielson, Nathan P.; Yin, Lichen; Guan, Linna; Chaudhury, Isthier; Lu, Hua

    2013-01-01

    Diblock copolymers consisting of poly(ethylene glycol)-block-poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-L-glutamate) (PEG-b-PVBLG-8) were synthesized and evaluated for their ability to mediate gene delivery in hard-to-transfect cells like IMR-90 human fetal lung fibroblasts and human embryonic stem cells (hESCs). The PEG-b-PVBLG-8 contained a membrane-disruptive, cationic, helical polypeptide block (PVBLG-8) for complexing with DNA and a hydrophilic PEG block to improve the biocompatibility of the gene delivery vehicle. The incorporation of PEG effectively reduced the toxicity of the helical PVBLG-8 block without dramatically compromising the polymer's ability to destabilize membranes or form complexes with DNA. PEG-b-PVBLG-8 copolymers with low (n = 76) and high (n = 287) degrees of polymerization (n) of the PVBLG-8 block were synthesized and evaluated for gene delivery. PEG-b-PVBLG-8 diblock polymers with a high degree of polymerization have a greater transfection efficiency and lower toxicity in IMR-90 cells than the commercial reagent Lipofectamine 2000. The usefulness of PEG-b-PVBLG-8 was further demonstrated via the successful transfection of hESCs without a measured loss in cell pluripotency markers. PMID:23997932

  16. Nucleofection Mediates High-Efficiency Stable Gene Knockdown and Transgene Expression in Human Embryonic Stem Cells

    PubMed Central

    Hohenstein, Kristi A.; Pyle, April D.; Chern, Jing Yi; Lock, Leslie F.; Donovan, Peter J.

    2013-01-01

    High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. PMID:18323409

  17. Erythroid activator NF-E2, TAL1 and KLF1 play roles in forming the LCR HSs in the human adult β-globin locus.

    PubMed

    Kim, Yea Woon; Yun, Won Ju; Kim, AeRi

    2016-06-01

    The β-like globin genes are developmental stage specifically transcribed in erythroid cells. The transcription of the β-like globin genes requires erythroid specific activators such as GATA-1, NF-E2, TAL1 and KLF1. However, the roles of these activators have not fully elucidated in transcription of the human adult β-globin gene. Here we employed hybrid MEL cells (MEL/ch11) where a human chromosome containing the β-globin locus is present and the adult β-globin gene is highly transcribed by induction. The roles of erythroid specific activators were analyzed by inhibiting the expression of NF-E2, TAL1 or KLF1 in MEL/ch11 cells. The loss of each activator decreased the transcription of human β-globin gene, locus wide histone hyperacetylation and the binding of other erythroid specific activators including GATA-1, even though not affecting the expression of other activators. Notably, sensitivity to DNase I was reduced in the locus control region (LCR) hypersensitive sites (HSs) with the depletion of activators. These results indicate that NF-E2, TAL1 and KLF1, all activators play a primary role in HSs formation in the LCR. It might contribute to the transcription of human adult β-globin gene by allowing the access of activators and cofactors. The roles of activators in the adult β-globin locus appear to be different from the roles in the early fetal locus. PMID:27026582

  18. Neofunctionalization of embryonic head patterning genes facilitates the positioning of novel traits on the dorsal head of adult beetles.

    PubMed

    Zattara, Eduardo E; Busey, Hannah A; Linz, David M; Tomoyasu, Yoshinori; Moczek, Armin P

    2016-07-13

    The origin and integration of novel traits are fundamental processes during the developmental evolution of complex organisms. Yet how novel traits integrate into pre-existing contexts remains poorly understood. Beetle horns represent a spectacular evolutionary novelty integrated within the context of the adult dorsal head, a highly conserved trait complex present since the origin of insects. We investigated whether otd1/2 and six3, members of a highly conserved gene network that instructs the formation of the anterior end of most bilaterians, also play roles in patterning more recently evolved traits. Using ablation-based fate-mapping, comparative larval RNA interference (RNAi) and transcript sequencing, we found that otd1/2, but not six3, play a fundamental role in the post-embryonic formation of the adult dorsal head and head horns of Onthophagus beetles. By contrast, neither gene appears to pattern the adult head of Tribolium flour beetles even though all are expressed in the dorsal head epidermis of both Onthophagus and Tribolium We propose that, at least in beetles, the roles of otd genes during post-embryonic development are decoupled from their embryonic functions, and that potentially non-functional post-embryonic expression in the dorsal head facilitated their co-option into a novel horn-patterning network during Onthophagus evolution. PMID:27412276

  19. High Expression of hTERT and Stemness Genes in BORIS/CTCFL Positive Cells Isolated from Embryonic Cancer Cells

    PubMed Central

    Alberti, Loredana; Renaud, Stéphanie; Losi, Lorena; Leyvraz, Serge; Benhattar, Jean

    2014-01-01

    BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3–5% of total). The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2) and cancer stem cell marker genes (CD44 and ALDH1) compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease. PMID:25279549

  20. Structure and Properties of a Bis-Histidyl Ligated Globin from Caenorhabditis elegans†

    PubMed Central

    Yoon, Jungjoo; Herzik, Mark A.; Winter, Michael B.; Tran, Rosalie; Olea, Charles; Marletta, Michael A.

    2010-01-01

    Globins are heme-containing proteins that are best known for their roles in oxygen (O2) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes have been recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O2 sensing behavior. Here, we describe GLB-6, a novel globin-like protein expressed in the neurons of C. elegans. Recombinantly expressed full-length GLB-6 contains a heme site with spectral features that are similar to those of other bis-histidyl ligated globins, such as neuroglobin and cytoglobin. In contrast to these globins, however, ligands such as CO, NO, and CN− do not bind to the heme in GLB-6, demonstrating that the endogenous histidine ligands are likely very tightly coordinated. Additionally, GLB-6 exhibits rapid two-state autoxidation kinetics in the presence of physiological O2 levels as well as a low redox potential (−193 ± 2 mV). A high resolution (1.40 Å) crystal structure of the ferric form of the heme-domain of GLB-6 confirms both the putative globin-fold and bis-histidyl ligation and also demonstrates key structural features that can be correlated with the unusual ligand binding and redox properties exhibited by the full-length protein. Taken together, the biochemical properties of GLB-6 suggest that this neural protein would most likely serve as a physiological sensor for O2 in C. elegans via redox signaling and/or electron transfer. PMID:20518498

  1. Polymorphic core promoter GA-repeats alter gene expression of the early embryonic developmental genes.

    PubMed

    Valipour, E; Kowsari, A; Bayat, H; Banan, M; Kazeminasab, S; Mohammadparast, S; Ohadi, M

    2013-12-01

    Protein complexes that bind to 'GAGA' DNA elements are necessary to replace nucleosomes to create a local chromatin environment that facilitates a variety of site-specific regulatory responses. Three to four elements are required for the disruption of a preassembled nucleosome. We have previously identified human protein-coding gene core promoters that are composed of exceptionally long GA-repeats. The functional implication of those GA-repeats is beginning to emerge in the core promoter of the human SOX5 gene, which is involved in multiple developmental processes. In the current study, we analyze the functional implication of GA-repeats in the core promoter of two additional genes, MECOM and GABRA3, whose expression is largely limited to embryogenesis. We report a significant difference in gene expression as a result of different alleles across those core promoters in the HEK-293 cell line. Across-species homology check for the GABRA3 GA-repeats revealed that those repeats are evolutionary conserved in mouse and primates (p<1 × 10(-8)). The MECOM core promoter GA-repeats are also conserved in numerous species, of which human has the longest repeat and complexity. We propose a novel role for GA-repeat core promoters to regulate gene expression in the genes involved in development and evolution. PMID:24055488

  2. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons

    PubMed Central

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S.

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP–GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP–GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP–GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin–GPR54 signaling, which could be a potential pathway to suppress cell migration. PMID:26973595

  3. Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells

    PubMed Central

    Bai, Yinshan; Feng, Meiying; Liu, Shanshan; Wei, Hengxi; Li, Li; Zhang, Xianwei; Shen, Chao; Zhang, Shouquan; Ma, Ningfang

    2016-01-01

    Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency-associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs. PMID:27353491

  4. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons.

    PubMed

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP-GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP-GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP-GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin-GPR54 signaling, which could be a potential pathway to suppress cell migration. PMID:26973595

  5. Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines

    PubMed Central

    Yamamizu, Kohei; Sharov, Alexei A.; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B.; Schlessinger, David; Ko, Minoru S. H.

    2016-01-01

    Mouse embryonic stem cells (ESCs) can differentiate into a wide range – and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this “NIA Mouse ESC Bank,” we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. PMID:27150017

  6. Homozygous Disruption of the Tip60 Gene Causes Early Embryonic Lethality

    PubMed Central

    Hu, Yaofei; Fisher, Joseph B.; Koprowski, Stacy; McAllister, Donna; Kim, Min-Su; Lough, John

    2009-01-01

    Tat-interactive protein 60 (Tip60) is a member of the MYST family, proteins in which are related by an atypical histone acetyltransferase (HAT) domain. Although Tip60 has been implicated in cellular activities including DNA repair, apoptosis and transcriptional regulation, its function during embryonic development is unknown. We ablated the Tip60 gene (Htatip) from the mouse by replacing exons 1–9 with a neomycin resistance cassette. Development and reproduction of wild-type and heterozygous animals was normal. However, homozygous ablation of the Tip60 gene caused embryolethality near the blastocyst stage of development, as evidenced by inability of cells in Tip60-null blastocysts to hatch and survive in culture. Monitoring cell proliferation and death by detecting EdU-substituted DNA and TUNEL labeling revealed suppression of cell proliferation concomitant with increased cell death as Tip60-null cells attempted to hatch from blastocysts. These findings indicate that Tip60 is essential for cellular survival during the blastocyst-gastrula transition of embryogenesis. PMID:19842187

  7. Differentially expressed genes in embryonic cardiac tissues of mice lacking Folr1 gene activity

    PubMed Central

    Zhu, Huiping; Cabrera, Robert M; Wlodarczyk, Bogdan J; Bozinov, Daniel; Wang, Deli; Schwartz, Robert J; Finnell, Richard H

    2007-01-01

    Background Heart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs), it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects [1-3]. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRα) gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis. Results We have observed cardiovascular abnormalities including outflow tract and aortic arch arterial defects in genetically compromised Folr1 knockout mice. In order to investigate the molecular mechanisms underlying the failure to complete development of outflow tract and aortic arch arteries in the Folr1 knockout mouse model, we examined tissue-specific gene expression difference between Folr1 nullizygous embryos and morphologically normal heterozygous embryos during early cardiac development (14-somite stage), heart tube looping (28-somite stage), and outflow track septation (38-somite stage). Microarray analysis was performed as a primary screening, followed by investigation using quantitative real-time PCR assays. Gene ontology analysis highlighted the following ontology groups: cell migration, cell motility and localization of cells, structural constituent of cytoskeleton, cell-cell adhesion, oxidoreductase, protein folding and mRNA processing. This study provided preliminary data and suggested potential candidate genes for further description and investigation. Conclusion The results suggested that Folr1 gene ablation and abnormal folate homeostasis altered gene expression in developing heart and conotruncal tissues. These changes affected normal cytoskeleton structures, cell migration and motility as well as cellular

  8. A randomized Phase I/II Trial of HQK-1001, an oral fetal globin gene inducer, in β–thalassaemia intermedia and HbE/β–thalassaemia

    PubMed Central

    Fucharoen, Suthat; Inati, Adlette; Siritanaratku, Noppadol; Thein, Swee Lay; Wargin, William C.; Koussa, Suzanne; Taher, Ali; Chaneim, Nattawara; Boosalis, Michael; Berenson, Ronald; Perrine, Susan P.

    2014-01-01

    β–thalassemia intermedia syndromes (BTI) cause hemolytic anemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anemia. Sodium 2,2 dimethylbutyrate (HQK-1001), an orally bioavailable short-chain fatty acid derivative, induces γ-globin expression experimentally and is well-tolerated in normal subjects. Accordingly, a randomized, blinded, placebo-controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/β0 thalassemia and 7 with β+/β0 thalassemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK-1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg/day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t1/2 of 10–12 hours. Adverse events with HQK-1001 treatment were not significantly different from placebo treatment. Median HbF increased with the 20 mg/kg treatment doses above baseline levels by 6.6% and 0.44 g/dL (p <0.01) in 8/9 subjects; total hemoglobin (Hgb) increased by a mean of 1.1 gm/dL in 4/9 subjects. These findings identify a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK-1001 with longer dosing to definitively evaluate its hematologic potential appears warranted. PMID:23530969

  9. Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation

    PubMed Central

    Pryzhkova, Marina V; Peters, Ann; Zambidis, Elias T

    2012-01-01

    Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and insitu immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research. PMID:20541472

  10. Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

    PubMed

    Krivega, Ivan; Byrnes, Colleen; de Vasconcellos, Jaira F; Lee, Y Terry; Kaushal, Megha; Dean, Ann; Miller, Jeffery L

    2015-07-30

    Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies. PMID:25979948

  11. Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell–Related Genes

    PubMed Central

    Bais, Manish; McLean, Jody; Sebastiani, Paola; Young, Megan; Wigner, Nathan; Smith, Temple; Kotton, Darrell N.; Einhorn, Thomas A.; Gerstenfeld, Louis C.

    2009-01-01

    Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture

  12. Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation.

    PubMed

    Lenka, Nibedita; Krishnan, Shruthi; Board, Philip; Rangasamy, Danny

    2014-06-01

    Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen. PMID:24610122

  13. Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions.

    PubMed Central

    Moon, A M; Ley, T J

    1990-01-01

    DNA sequences located in a region 6-18 kilobases (kb) upstream from the human epsilon-globin gene are known as the locus-activating region (LAR) or dominant control region. This region is thought to play a key role in chromatin organization of the beta-like globin gene cluster during erythroid development. The beta-globin LAR activates linked globin genes in transiently or stably transfected erythroleukemia cells and in erythroid cells of transgenic mice. Since the human beta-globin LAR is functional in mice, we reasoned that critical LAR sequence elements might be conserved between mice and humans. We therefore cloned murine genomic sequences homologous to one portion of the human LAR (site II, positions -11,054 to -10,322 with respect to the human epsilon gene). We found that this murine DNA fragment (mouse LAR site II) and sequences homologous to human LAR sites I and III are located upstream from the mouse beta-like globin gene cluster and determined that their locations relative to the cluster are similar to that of their human counterparts. The homologous site II sequences are 70% identical between mice and humans over a stretch of approximately 800 base pairs. Multiple core sequences with greater than 80% identity were present within this region. Transient and stable transfection assays of K562 erythroleukemia cells demonstrated that both human and mouse LAR elements contain enhancer activity and confer hemin inducibility on a linked human gamma-globin promoter. These results suggest that primary structural elements--and the spatial organization of these elements--are important for function of the beta-globin LAR. Images PMID:2217202

  14. Genome-wide gene expression analysis of mouse embryonic stem cells exposed to p-dichlorobenzene.

    PubMed

    Tani, Hidenori; Takeshita, Jun-Ichi; Aoki, Hiroshi; Abe, Ryosuke; Toyoda, Akinobu; Endo, Yasunori; Miyamoto, Sadaaki; Gamo, Masashi; Torimura, Masaki

    2016-09-01

    Because of the limitations of whole animal testing approaches for toxicological assessment, new cell-based assay systems have been widely studied. In this study, we focused on two biological products for toxicological assessment: mouse embryonic stem cells (mESCs) and long noncoding RNAs (lncRNAs). mESCs possess the abilities of self-renewal and differentiation into multiple cell types. LlncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to chemicals. We exposed mESCs to p-dichlorobenzene (p-DCB) for 1 or 28 days (daily dose), extracted total RNA, and performed deep sequencing analyses. The genome-wide gene expression analysis indicated that mechanisms modulating proteins occurred following acute and chronic exposures, and mechanisms modulating genomic DNA occurred following chronic exposure. Moreover, our results indicate that three novel lncRNAs (Snora41, Gm19947, and Scarna3a) in mESCs respond to p-DCB exposure. We propose that these lncRNAs have the potential to be surrogate indicators of p-DCB responses in mESCs. PMID:26975756

  15. Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels.

    PubMed

    Yelin, Ronit; Schyr, Racheli Ben-Haroush; Kot, Hadas; Zins, Sharon; Frumkin, Ayala; Pillemer, Graciela; Fainsod, Abraham

    2005-03-01

    Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental malformations caused by alcohol consumption during pregnancy. Fetal Alcohol Syndrome (FAS), the strongest manifestation of FASD, results in short stature, microcephally and facial dysmorphogenesis including microphthalmia. Using Xenopus embryos as a model developmental system, we show that ethanol exposure recapitulates many aspects of FAS, including a shortened rostro-caudal axis, microcephally and microphthalmia. Temporal analysis revealed that Xenopus embryos are most sensitive to ethanol exposure between late blastula and early/mid gastrula stages. This window of sensitivity overlaps with the formation and early function of the embryonic organizer, Spemann's organizer. Molecular analysis revealed that ethanol exposure of embryos induces changes in the domains and levels of organizer-specific gene expression, identifying Spemann's organizer as an early target of ethanol. Ethanol also induces a defect in convergent extension movements that delays gastrulation movements and may affect the overall length. We show that mechanistically, ethanol is antagonistic to retinol (Vitamin A) and retinal conversion to retinoic acid, and that the organizer is active in retinoic acid signaling during early gastrulation. The model suggests that FASD is induced in part by an ethanol-dependent reduction in retinoic acid levels that are necessary for the normal function of Spemann's organizer. PMID:15708568

  16. A novel embryonic plasticity gene signature that predicts metastatic competence and clinical outcome

    PubMed Central

    Soundararajan, Rama; Paranjape, Anurag N.; Barsan, Valentin; Chang, Jeffrey T.; Mani, Sendurai A.

    2015-01-01

    Currently, very few prognosticators accurately predict metastasis in cancer patients. In order to complete the metastatic cascade and successfully colonize distant sites, carcinoma cells undergo dynamic epithelial-mesenchymal-transition (EMT) and its reversal, mesenchymal-epithelial-transition (MET). While EMT-centric signatures correlate with response to therapy, they are unable to predict metastatic outcome. One reason is due to the wide range of transient phenotypes required for a tumor cell to disseminate and recreate a similar histology at distant sites. Since such dynamic cellular processes are also seen during embryo development (epithelial-like epiblast cells undergo transient EMT to generate the mesoderm, which eventually redifferentiates into epithelial tissues by MET), we sought to utilize this unique and highly conserved property of cellular plasticity to predict metastasis. Here we present the identification of a novel prognostic gene expression signature derived from mouse embryonic day 6.5 that is representative of extensive cellular plasticity, and predicts metastatic competence in human breast tumor cells. This signature may thus complement conventional clinical parameters to offer accurate prediction for outcome among multiple classes of breast cancer patients. PMID:26123483

  17. Disruption of the Mouse mTOR Gene Leads to Early Postimplantation Lethality and Prohibits Embryonic Stem Cell Development

    PubMed Central

    Gangloff, Yann-Gaël; Mueller, Matthias; Dann, Stephen G.; Svoboda, Petr; Sticker, Melanie; Spetz, Jean-Francois; Um, Sung Hee; Brown, Eric J.; Cereghini, Silvia; Thomas, George; Kozma, Sara C.

    2004-01-01

    The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR+/−) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR+/− mice, embryonic development of homozygous mTOR−/− mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR−/− embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR−/− embryos. PMID:15485918

  18. Pluripotent embryonic stem cells and multipotent adult germline stem cells reveal similar transcriptomes including pluripotency-related genes.

    PubMed

    Meyer, S; Nolte, J; Opitz, L; Salinas-Riester, G; Engel, W

    2010-11-01

    DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs). PMID:20624824

  19. Analysis of β-globin chromatin micro-environment using a novel 3C variant, 4Cv.

    PubMed

    Pink, Ryan C; Eskiw, Christopher H; Caley, Daniel P; Carter, David R F

    2010-01-01

    Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C) have provided us with the tools to probe chromosomal structures. These techniques have been applied to the β-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of β-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv), which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the β-globin locus in mice and show that a significant proportion of the interactions of β-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, β-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the β-globin locus leads to a change in nuclear position relative to the chromosome territory. PMID:20927371

  20. Cationic star copolymers based on β-cyclodextrins for efficient gene delivery to mouse embryonic stem cell colonies.

    PubMed

    Loh, Xian Jun; Wu, Yun-Long

    2015-07-11

    A cationic star copolymer with a β-cyclodextrin core was developed for nonviral gene transfer to mouse embryonic stem cells (mESCs). The copolymer comprises poly(2-dimethyl aminoethyl methacrylate) as the cationic component and poly(2-hydroxyethyl methacrylate) as the non-toxic stealth component. These materials have very low toxicity and show highly efficient transfection to mESC colonies. PMID:26040469

  1. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene.

    PubMed

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-03-01

    The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345970

  2. Compounds of the anthracycline family of antibiotics elevate human gamma-globin expression both in erythroid cultures and in a transgenic mouse model.

    PubMed

    Spyrou, Pandelis; Phylactides, Marios; Lederer, Carsten W; Kithreotis, Lucas; Kirri, Andriani; Christou, Soteroulla; Kkolou, Elena; Kanavakis, Emanuel; Anagnou, Nicholas P; Stamatoyannopoulos, George; Kleanthous, Marina

    2010-01-01

    We examined the effect of the anthracyclines aclarubicin, bleomycin, daunorubicin, doxorubicin and idarubicin on human gamma- and beta-globin promoter activity in an in vitro luciferase assay, ex vivo in erythroid cultures and in vivo in transgenic mice carrying the human gamma-globin gene. Effects in erythroid liquid cultures derived from healthy donors were assayed by evaluating HbF production with high performance liquid chromatography and by measuring mRNA levels of the globin genes and the proportion of erythroblasts containing HbF. Compounds testing positive in the in vitro and ex vivo assays were applied to erythroid cultures derived from thalassaemic patients. Doxorubicin, idarubicin and daunorubicin increased HbF production in cultures of both, healthy and thalassaemic donors. Daunorubicin induced HbF in thalassaemic cells ex vivo with the highest statistical significance and, importantly and in contrast to the clinical HbF inducer hydroxyurea, showed specific induction of gamma-globin without associated induction of alpha-globin. Daunorubicin was screened in transgenic mice carrying the human (A)gamma-globin gene, and it resulted in increased (A)gamma-globin mRNA levels. Our results indicate that anthracyclines are a promising group of compounds with the potential to provide lead substances for the synthesis of new agents with clinical applications as gamma-globin gene inducers. In parallel, future studies of the epigenetic effects of the five anthracyclines on the beta-globin locus will generate possible mechanistic leads on the regulation of the globin genes. PMID:19914848

  3. Ascl1 is a required downstream effector of Gsx gene function in the embryonic mouse telencephalon

    PubMed Central

    Wang, Bei; Waclaw, Ronald R; Allen, Zegary J; Guillemot, Francois; Campbell, Kenneth

    2009-01-01

    Background The homeobox gene Gsx2 (formerly Gsh2) is known to regulate patterning in the lateral ganglionic eminence (LGE) of the embryonic telencephalon. In its absence, the closely related gene Gsx1 (previously known as Gsh1) can partially compensate in the patterning and differentiation of ventral telencephalic structures, such as the striatum. However, the cellular and molecular mechanisms underlying this compensation remain unclear. Results We show here that in the Gsx2 mutants Gsx1 is expressed in only a subset of the ventral telencephalic progenitors that normally express Gsx2. Based on the similarities in the expression of Gsx1 and Ascl1 (Mash1) within the Gsx2 mutant LGE, we examined whether Ascl1 plays an integral part in the Gsx1-based recovery. Ascl1 mutants show only modest alterations in striatal development; however, in Gsx2;Ascl1 double mutants, striatal development is severely affected, similar to that seen in the Gsx1;Gsx2 double mutants. This is despite the fact that Gsx1 is expressed, and even expands, in the Gsx2;Ascl1 mutant LGE, comparable to that seen in the Gsx2 mutant. Finally, Notch signaling has recently been suggested to be required for normal striatal development. In spite of the fact that Notch signaling is severely disrupted in Ascl1 mutants, it actually appears to be improved in the Gsx2;Ascl1 double mutants. Conclusion These results, therefore, reveal a non-proneural requirement of Ascl1 that together with Gsx1 compensates for the loss of Gsx2 in a subset of LGE progenitors. PMID:19208224

  4. Functional equivalence of Hox gene products in the specification of the tritocerebrum during embryonic brain development of Drosophila.

    PubMed

    Hirth, F; Loop, T; Egger, B; Miller, D F; Kaufman, T C; Reichert, H

    2001-12-01

    Hox genes encode evolutionarily conserved transcription factors involved in the specification of segmental identity during embryonic development. This specification of identity is thought to be directed by differential Hox gene action, based on differential spatiotemporal expression patterns, protein sequence differences, interactions with co-factors and regulation of specific downstream genes. During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere; in the absence of labial, the cells in this brain region do not acquire a neuronal identity and major axonal pathfinding deficits result. We have used genetic rescue experiments to investigate the functional equivalence of the Drosophila Hox gene products in the specification of the tritocerebral neuromere. Using the Gal4-UAS system, we first demonstrate that the labial mutant brain phenotype can be rescued by targeted expression of the Labial protein under the control of CNS-specific labial regulatory elements. We then show that under the control of these CNS-specific regulatory elements, all other Drosophila Hox gene products, except Abdominal-B, are able to efficiently replace Labial in the specification of the tritocerebral neuromere. We also observe a correlation between the rescue efficiency of the Hox proteins and the chromosomal arrangement of their encoding loci. Our results indicate that, despite considerably diverged sequences, most Hox proteins are functionally equivalent in their ability to replace Labial in the specification of neuronal identity. This suggests that in embryonic brain development, differences in Hox gene action rely mainly on cis-acting regulatory elements and not on Hox protein specificity. PMID:11731458

  5. Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells.

    PubMed

    Bai, Yinshan; Feng, Meiying; Liu, Shanshan; Wei, Hengxi; Li, Li; Zhang, Xianwei; Shen, Chao; Zhang, Shouquan; Ma, Ningfang

    2016-08-01

    Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency‑associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs. PMID:27353491

  6. siDNMT1 Increases γ-globin Expression in Chemical-Inducer-of-Dimerization (CID)-Dependent Mouse βYAC Bone Marrow Cells and in Baboon Erythroid Progenitor Cell Cultures

    PubMed Central

    Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; Ruiz, Maria Armila; Peterson, Kenneth; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult stage erythroid cells. 2) Methods DNMT1 levels were reduced by nucleofection of siRNA targeting DNMT1 in chemical-inducer-of-dimerization (CID)-dependent multipotential mouse bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and in primary cultures of erythroid progenitor cells derived from CD34+ baboon BM cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real time PCR and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabelling of globin chains followed by HPLC analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. 3) Results Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin mRNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. 4) Conclusion DNMT1 is required to maintain DNA methylation of the γ-globin gene promoters and repress γ-globin gene expression in adult-stage erythroid cells. PMID:20974210

  7. Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts

    PubMed Central

    Marthandan, Shiva; Priebe, Steffen; Baumgart, Mario; Groth, Marco; Cellerino, Alessandro; Guthke, Reinhard; Hemmerich, Peter; Diekmann, Stephan

    2015-01-01

    Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin. PMID:26339636

  8. Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts.

    PubMed

    Marthandan, Shiva; Priebe, Steffen; Baumgart, Mario; Groth, Marco; Cellerino, Alessandro; Guthke, Reinhard; Hemmerich, Peter; Diekmann, Stephan

    2015-01-01

    Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin. PMID:26339636

  9. Gene Expression Profile of Adult Human Olfactory Bulb and Embryonic Neural Stem Cell Suggests Distinct Signaling Pathways and Epigenetic Control

    PubMed Central

    Marei, Hany E. S.; Ahmed, Abd-Elmaksoud; Michetti, Fabrizio; Pescatori, Mario; Pallini, Roberto; Casalbore, Patricia; Cenciarelli, Carlo; Elhadidy, Mohamed

    2012-01-01

    Global gene expression profiling was performed using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define a gene expression pattern and signaling pathways that are specific for each cell lineage. We have demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. By Gene Ontology, 325 out of 3875 investigated gene sets were scientifically different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison had revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR that are known to be involved in NS cell fates determination. The transcriptional signature also deciphers the role of genes involved in epigenetic modifications. SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, were found specifically up-regulated in our OBNSC but not in hENSC. Differences in gene expression profile of transcripts controlling epigenetic modifications, and signaling pathways might indicate differences in the therapeutic potential of our examined two cell populations in relation to in cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults. PMID:22485144

  10. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

    PubMed

    Aimola, Idowu A; Inuwa, Hajiya M; Nok, Andrew J; Mamman, Aisha I; Bieker, James J

    2016-04-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer. PMID:26879870

  11. Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

    PubMed Central

    Quan, Yuan; Yan, Qing; Morales, John E.

    2015-01-01

    Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells

  12. Embryonic Atrazine Exposure Elicits Alterations in Genes Associated with Neuroendocrine Function in Adult Male Zebrafish.

    PubMed

    Wirbisky, Sara E; Sepúlveda, Maria S; Weber, Gregory J; Jannasch, Amber S; Horzmann, Katharine A; Freeman, Jennifer L

    2016-09-01

    The developmental origins of health and disease (DOHaD) hypothesis states that exposure to environmental stressors early in life can elicit genome and epigenome changes resulting in an increased susceptibility of a disease state during adulthood. Atrazine, a common agricultural herbicide used throughout the Midwestern United States, frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. In our previous studies, zebrafish was exposed to 0, 0.3, 3, or 30 parts per billion (μg/l) atrazine through embryogenesis, rinsed, and allowed to mature to adulthood. A decrease in spawning was observed with morphological alterations in offspring. In addition, adult females displayed an increase in ovarian progesterone and follicular atresia, alterations in levels of a serotonin metabolite and serotonin turnover in brain tissue, and transcriptome changes in brain and ovarian tissue supporting neuroendocrine alterations. As reproductive dysfunction is also influenced by males, this study assessed testes histology, hormone levels, and transcriptomic profiles of testes and brain tissue in the adult males. The embryonic atrazine exposure resulted in no alterations in body or testes weight, gonadosomatic index, testes histology, or levels of 11-ketotestosterone or testosterone. To further investigate potential alterations, transcriptomic profiles of adult male testes and brain tissue was completed. This analysis demonstrated alterations in genes associated with abnormal cell and neuronal growth and morphology; molecular transport, quantity, and production of steroid hormones; and neurotransmission with an emphasis on the hypothalamus-pituitary-adrenal and hypothalamus-pituitary-thyroid axes. Overall, this data indicate future studies should focus on additional neuroendocrine endpoints to determine potential functional impairments. PMID:27413107

  13. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.

    PubMed Central

    Askew, G R; Doetschman, T; Lingrel, J B

    1993-01-01

    Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders. Images PMID:8391633

  14. C. elegans SoxB genes are dispensable for embryonic neurogenesis but required for terminal differentiation of specific neuron types

    PubMed Central

    Vidal, Berta; Santella, Anthony; Serrano-Saiz, Esther; Bao, Zhirong; Chuang, Chiou-Fen; Hobert, Oliver

    2015-01-01

    Neurogenesis involves deeply conserved patterning molecules, such as the proneural basic helix-loop-helix transcription factors. Sox proteins and specifically members of the SoxB and SoxC groups are another class of conserved transcription factors with an important role in neuronal fate commitment and differentiation in various species. In this study, we examine the expression of all five Sox genes of the nematode C. elegans and analyze the effect of null mutant alleles of all members of the SoxB and SoxC groups on nervous system development. Surprisingly, we find that, unlike in other systems, neither of the two C. elegans SoxB genes sox-2 (SoxB1) and sox-3 (SoxB2), nor the sole C. elegans SoxC gene sem-2, is broadly expressed throughout the embryonic or adult nervous system and that all three genes are mostly dispensable for embryonic neurogenesis. Instead, sox-2 is required to maintain the developmental potential of blast cells that are generated in the embryo but divide only postembryonically to give rise to differentiated neuronal cell types. Moreover, sox-2 and sox-3 have selective roles in the terminal differentiation of specific neuronal cell types. Our findings suggest that the common themes of SoxB gene function across phylogeny lie in specifying developmental potential and, later on, in selectively controlling terminal differentiation programs of specific neuron types, but not in broadly controlling neurogenesis. PMID:26153233

  15. Signaling mechanisms for the activation of an embryonic gene program during the hypertrophy of cardiac ventricular muscle.

    PubMed

    Chien, R

    1992-01-01

    To study the signaling mechanisms which mediate ventricular hypertrophy, we utilized the induction of the ANF gene as a marker of the hypertrophic response. The induction of the atrial natriuretic factor gene (ANF) is one of the most conserved features of ventricular hypertrophy, occurring in multiple species (mouse, rat, hamster, canine, and human) in response to diverse stimuli (hormonal, mechanical, pressure/volume overload, genetic, IHSS, hypertension, etc.). The ANF gene is expressed in both the atrial and ventricular compartments during embryonic development, but shortly after birth ANF expression is down-regulated to negligible levels in the adult myocardium. Since the reactivation of ANF gene expression in the hypertrophied ventricle is a hallmark of the activation of an embryonic gene program, it has also become of interest to determine if similar mechanisms activate ANF expression during hypertrophy and the initial stages of cardiogenesis. A combination of cotransfection, microinjection, and transgenic approaches has been coupled to well characterized cultured cell systems and in vivo murine models employing normal and transgenic mice. The microinjection of oncogenic RAS proteins into living myocardial cells does not lead to the activation of cell proliferation, but activates ANF gene expression, as assessed by immunofluorescence. Co-transfection of mutant and wild-type RAS expression vectors with a ANF-luciferase fusion gene supports a direct effect of activated RAS on ANF gene transcription. Co-transfection of a dominant negative RAS vector effectively inhibits the induction of the ANF gene during alpha adrenergic mediated hypertrophy of ventricular muscle cells, thereby establishing that a RAS-mediated pathway is required for ANF induction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1299210

  16. Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis

    PubMed Central

    Guo, Guoji; Pinello, Luca; Han, Xiaoping; Lai, Shujing; Shen, Li; Lin, Ta-Wei; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

    2015-01-01

    Summary Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. We define highly variable gene clusters with distinct chromatin states; and show that bivalent genes are prone to expression variation. At the same time, we identify an ESC priming pathway that initiates the exit from the naïve ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum free culture reduces cellular heterogeneity and transcriptome variation in ESCs. PMID:26804902

  17. Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

    SciTech Connect

    Theunissen, P.T.; Robinson, J.F.; Pennings, J.L.A.; Herwijnen, M.H. van; Kleinjans, J.C.S.; Piersma, A.H.

    2012-08-01

    Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24 h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

  18. Beta-Globin Gene Haplotypes Among Cameroonians and Review of the Global Distribution: Is There a Case for a Single Sickle Mutation Origin in Africa?

    PubMed Central

    Bitoungui, Valentina J. Ngo; Pule, Gift D.; Hanchard, Neil; Ngogang, Jeanne

    2015-01-01

    Abstract Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n=799) and Cameroon (19%; n=207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation. PMID:25748438

  19. Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

    PubMed

    Gelderman, Monique P; Baek, Jin Hyen; Yalamanoglu, Ayla; Puglia, Michele; Vallelian, Florence; Burla, Bo; Vostal, Jaroslav; Schaer, Dominik J; Buehler, Paul W

    2015-05-01

    Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice. PMID:25616571

  20. Beta-globin gene haplotypes among cameroonians and review of the global distribution: is there a case for a single sickle mutation origin in Africa?

    PubMed

    Bitoungui, Valentina J Ngo; Pule, Gift D; Hanchard, Neil; Ngogang, Jeanne; Wonkam, Ambroise

    2015-03-01

    Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n = 799) and Cameroon (19%; n = 207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation. PMID:25748438

  1. Differential regulation of osteogenic marker gene expression by Wnt-3a in embryonic mesenchymal multipotential progenitor cells.

    PubMed

    Derfoul, Assia; Carlberg, Alyssa L; Tuan, Rocky S; Hall, David J

    2004-06-01

    The Wnt family of secreted glycoproteins plays an integral role in embryonic development and differentiation. To explore the role of Wnt's in one aspect of differentiation, namely osteogenesis, we employed a retroviral gene transfer approach to express Wnt-3a in the multipotent murine embryonic mesenchymal cell line C3H10T1/2. We found that expression of Wnt-3a in these cells had a significant, positive effect on cell growth in serum-containing medium, in that the cells grew to very high densities compared to the control cells. Additionally, apoptosis was markedly inhibited by Wnt-3a. However, when the cells were grown in serum-deficient medium, the Wnt-3a-expressing cells arrested efficiently in G1 phase, indicating that serum growth factors were needed in addition to Wnt-3a for enhanced proliferation. Wnt-3a-expressing cells exhibited high levels of alkaline phosphatase gene expression and enzymatic activity, but did not show any matrix mineralization. Unexpectedly, basal expression of bone sialoprotein, osteocalcin, and osteopontin were markedly inhibited by Wnt-3a, as were other known target genes of Wnt-3a, such as Brachyury, FGF-10, and Cdx1. When Wnt-3a-expressing cells were treated with osteogenic supplements in the presence of BMP-2, alkaline phosphatase gene expression and activity were further elevated. Additionally, BMP-2 was able to reverse the inhibitory effect of Wnt-3a on osteocalcin and osteopontin gene expression. These results indicate that while Wnt-3a represses basal expression of some osteogenic genes, this repression can be partially reversed by BMP-2. Finally, the enhanced gene expression of alkaline phosphatase induced by Wnt-3a could be effectively suppressed by the combined action of dexamethasone and 1,25-dihydroxyvitamin D(3). These data show for the first time that Wnt-3a has an unusual effect on multipotential embryonic cells, in that it enhances cellular proliferation and expression of alkaline phosphatase, while it represses most

  2. Characterization of Clinical and Laboratory Profiles of the Deletional α2-Globin Gene Polyadenylation Signal Sequence (AATAAA > AATA- -) in an Indian Population.

    PubMed

    Deshpande, Prashant; Kamalanathan, Neelagandan; Sampath, Eswari; George, Biju; Shaji, Ramachandran V; Edison, Eunice S

    2015-01-01

    α-Thalassemia (α-thal) is characterized by large deletions involving the variable regions of α2 and/or α1 genes. Nondeletional mutations and polyadenylation (polyA) signal sequence motif mutations are less common. In this retrospective study, we describe a fragment length analysis-based polymerase chain reaction (PCR) assay for screening the T(Indian) (AATAAA > AATA- -; HBA2: c.*93_*94delAA) polyA signal deletion along with its clinical and laboratory presentation in 21 patients. Most of the patients were diagnosed in early adulthood with a clinical presentation ranging from asymptomatic in the heterozygous state to severe Hb H disease with a prominent hemolytic component in the homozygous state. On genetic analysis, 14 patients were found to be homozygotes, five were compound heterozygotes and two were heterozygotes. Thus, the T(Indian) polyA signal deletion is common in the Indian population and should be screened for in patients with nondeletional α-thal mutations. PMID:26365411

  3. A New Intergenic α-Globin Deletion (α-α(Δ125)) Found in a Kabyle Population.

    PubMed

    Rabbind Singh, Amrathlal; Lacan, Philippe; Cadet, Estelle; Bignet, Patricia; Dumesnil, Cécile; Vannier, Jean-Pierre; Joly, Philippe; Rochette, Jacques

    2016-03-01

    We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact. PMID:26911300

  4. Effect of AGM and fetal liver-derived stromal cell lines on globin expression in adult baboon (P. anubis) bone marrow-derived erythroid progenitors.

    PubMed

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  5. Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

    PubMed Central

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  6. Effect of temperature on gene expression of the Gh/Igf system during embryonic development in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Gabillard, Jean-Charles; Rescan, Pierre-Yves; Fauconneau, Benoit; Weil, Claudine; Le Bail, Pierre-Yves

    2003-08-01

    In fish, the GH/IGF system installs very early during development suggesting that this system could promote embryonic growth and development. In contrast to mammals, the embryonic growth rate of poikilotherms depends considerably on the incubation temperature. Therefore, the aim of this study was to determine if variations of embryo growth in response to temperature could be associated with modifications in the gene expression of the GH/IGF system. In this study, using whole mount in situ hybridisation, we demonstrated that embryo incubation temperature (4, 8, and 12 degrees C) did not change the timing of GH-1 and GH-2 mRNA expression in somatotroph cells (stage 24). Similarly, at hatching (stage 30), we did not observe an obvious difference in GH protein and GH-1 and GH-2 transcript amounts in relation to the incubation temperature. Furthermore, from stage 22 to 25, the highest temperature led to a specific up-regulation of IGF-2 (2-fold between 4 and 12 degrees C), and both IGF-RIa and IGFRIb mRNA (1.5-fold between 4 and 12 degrees C), while no difference was observed for IGF-1 mRNA. Conversely, at hatching, the highest temperature specifically down-regulated IGF-2 (3-fold between 4 and 12 degrees C) and both IGF receptor mRNAs (2 fold between 4 and 12 degrees C) present in the head, while no difference was observed in the trunk. Our results demonstrated that different incubation temperatures during trout embryonic development did not change the stage of somatotroph cell appearance. Before hatching, IGF-2 and both IGF receptors, but not IGF-1 mRNA, were specifically up-regulated by high temperatures and could be related to the enhancement of embryonic growth rate. PMID:12884275

  7. A murine AP-endonuclease gene-targeted deficiency with post-implantation embryonic progression and ionizing radiation sensitivity.

    PubMed

    Ludwig, D L; MacInnes, M A; Takiguchi, Y; Purtymun, P E; Henrie, M; Flannery, M; Meneses, J; Pedersen, R A; Chen, D J

    1998-10-21

    Apurinic/apyrimidinic endonuclease (here designated APE/REF) carries out repair incision at abasic or single-strand break damages in mammals. This multifunctional protein also has putative role(s) as a cysteine 'reducing factor' (REF) in cell-stress transcriptional responses. To assess the significance of APE/REF for embryonic teratogenesis we constructed a more precisely targeted Ape/Ref-deficient genotype in mice. Ape/Ref gene replacement in ES cells eliminated the potential of APE/REF protein synthesis while retaining the Ape/Ref bi-directional promoter that avoided potential inactivation of an upstream gene. Chimeric animals crossed into Tac:N:NIHS-BC produced germline transmission. Homozygous null Ape/Ref-embryos exhibited successful implantation and nearly normal developmental progression until embryonic day 7.5 followed by morphogenetic failure and adsorption of embryos by day 9.5. We characterized the cellular events proceeding to embryonic lethality and examined ionizing radiation sensitivity of pre-implantation Ape/Ref-null embryos. After intermating of heterozygotes, Mendelian numbers of putative Ape/Ref-null progeny embryos at day 6.5 displayed a several-fold elevation of pycnotic, fragmenting cell nuclei within the embryo proper-the epiblast. Increased cell-nucleus degeneration occurred within epiblast cells while mitosis continued and before obvious morphogenetic disruption. Mitogenic response to epiblast cell death, if any, was ineffective for replacement of lost cells. Extra-embryonic yolk sac, a trophectoderm derived lineage retained normal appearance to day 9. Explanted homozygous Ape/Ref-null blastocysts displayed increased sensitivity to gamma-irradiation, most likely a manifestation of APE/REF incision defect. Our study establishes that this new Ape/Ref deficiency genotype is definitely capable of post-implantation developmental progression to the onset of gastrulation. Function(s) of APE/REF in base damage incision and also conceivably in

  8. Pleiotropic derepression of developmentally regulated cellular and viral genes by c-myc protooncogene products in undifferentiated embryonal carcinoma cells.

    PubMed Central

    Onclercq, R; Lavenu, A; Cremisi, C

    1989-01-01

    We show here in mouse embryonal carcinoma (EC) cells that the endo A gene is negatively regulated and shares negative transacting factors with the Py and SV40 viruses. The products of the proto-oncogene c-myc derepress at the transcriptional level the appropriately initiated expression of the endo A gene and activate the Py early promoter in EC stem cells. C-myc products also activate the endo A and the Py early promoters in TDM epithelial cells, and the Py early promoter in 3T6 cells in which the two genes are already expressed or can be expressed. Furthermore we show that the myc exon 1 is essential for activation and that this activation might be mediated by AP1 family factors. Images PMID:2536923

  9. Generation of KCL017 research grade human embryonic stem cell line carrying a mutation in VHL gene.

    PubMed

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-03-01

    The KCL017 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel-Lindau tumor suppressor E3 ubiquitin protein ligase (676+3A>T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345980

  10. Generation of KCL016 research grade human embryonic stem cell line carrying a mutation in VHL gene.

    PubMed

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL016 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel-Lindau tumor suppressor E3 ubiquitin protein ligase (676+3A>T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345783

  11. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  12. Generation of KCL036 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL036 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (38 trinucleotide repeats; 14 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  13. Generation of KCL012 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL012 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (46 trinucleotide repeats; 17 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  14. Generation of KCL027 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL027 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  15. Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

    EPA Science Inventory

    Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 μM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post...

  16. The relative cellular levels of CP2a and CP2b potentiates erythroid cell-specific expression of the {alpha}-globin gene by regulating the nuclear localization of CP2c

    SciTech Connect

    Chae, Ji Hyung; Kang, Ho Chul; Kim, Chul Geun

    2009-03-20

    CP2b activates {alpha}-globin expression in an erythroid cell-specific manner, through interaction with CP2c and PIAS1. Although CP2a is identical to CP2b except for lacking an exon encoding additional 36 amino acids and has the intrinsic DNA binding and transactivation properties, it does not exert any role in {alpha}-globin expression. Investigation of subcellular localization of exogenous CP2 proteins revealed that CP2a and CP2b were exclusively localized in the cytosol and nucleus, respectively. The CP2b-specific exon was in charge of the nuclear localization of CP2b. Interestingly, subcellular localization of CP2c was either in the nucleus or cytosol depending on the relative level of CP2a and CP2b although CP2c intrinsically localized in the cytosol in the absence of CP2a/CP2b. Finally, dramatic increment of hemoglobin expression was correlated with nuclear translocation of CP2c during MEL cell differentiation. Our data suggest that CP2b potentiate erythroid cell-specific {alpha}-globin expression by recruiting CP2c into the nucleus.

  17. Transcriptional Profiling Identifies Location-Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos

    PubMed Central

    Zhang, Rong-Ping; Liu, He-He; Liu, Jun-Ying; Hu, Ji-Wei; Yan, Xi-Ping; Wang, Ding-Min-Cheng; Li, Liang; Wang, Ji-Wen

    2015-01-01

    Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our

  18. In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor's Production.

    PubMed

    Fauzi, Iliana; Panoskaltsis, Nicki; Mantalaris, Athanasios

    2016-01-01

    Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs. PMID:26160454

  19. Maternal Experience with Predation Risk Influences Genome-Wide Embryonic Gene Expression in Threespined Sticklebacks (Gasterosteus aculeatus)

    PubMed Central

    Mommer, Brett C.; Bell, Alison M.

    2014-01-01

    There is growing evidence for nongenetic effects of maternal experience on offspring. For example, previous studies have shown that female threespined stickleback fish (Gasterosteus aculeatus) exposed to predation risk produce offspring with altered behavior, metabolism and stress physiology. Here, we investigate the effect of maternal exposure to predation risk on the embryonic transcriptome in sticklebacks. Using RNA-sequencing we compared genome-wide transcription in three day post-fertilization embryos of predator-exposed and control mothers. There were hundreds of differentially expressed transcripts between embryos of predator-exposed mothers and embryos of control mothers including several non-coding RNAs. Gene Ontology analysis revealed biological pathways involved in metabolism, epigenetic inheritance, and neural proliferation and differentiation that differed between treatments. Interestingly, predation risk is associated with an accelerated life history in many vertebrates, and several of the genes and biological pathways that were identified in this study suggest that maternal exposure to predation risk accelerates the timing of embryonic development. Consistent with this hypothesis, embryos of predator-exposed mothers were larger than embryos of control mothers. These findings point to some of the molecular mechanisms that might underlie maternal effects. PMID:24887438

  20. The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila.

    PubMed Central

    Song, Ho-Juhn; Billeter, Jean-Christophe; Reynaud, Enrique; Carlo, Troy; Spana, Eric P; Perrimon, Norbert; Goodwin, Stephen F; Baker, Bruce S; Taylor, Barbara J

    2002-01-01

    The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. We have examined fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. Our results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS. PMID:12524343

  1. Genomewide approaches for BACH1 target genes in mouse embryonic fibroblasts showed BACH1-Pparg pathway in adipogenesis.

    PubMed

    Matsumoto, Mitsuyo; Kondo, Keiichi; Shiraki, Takuma; Brydun, Andrey; Funayama, Ryo; Nakayama, Keiko; Yaegashi, Nobuo; Katagiri, Hideki; Igarashi, Kazuhiko

    2016-06-01

    The transcription repressor BTB and CNC homology 1 (BACH1) represses genes involved in heme metabolism and oxidative stress response. BACH1 also suppresses the p53-dependent cellar senescence in primary mouse embryonic fibroblasts (MEFs). To investigate the role of BACH1 in MEF other than its known functions, we carried out a genomewide mapping of binding site for BACH1 and its heterodimer partner MAFK in immortalized MEFs (iMEFs) using chromatin immunoprecipitation and next-generation sequencing technology (ChIP-sequence). The comparative analysis of the ChIP-sequence data and DNA microarray data from Bach1-deficient and wild-type (WT) iMEF showed 35 novel candidate target genes of BACH1. Among these genes, five genes (Pparg, Nfia, Ptplad2, Adcy1 and Ror1) were related with lipid metabolism. Bach1-deficient iMEFs showed increased expression of mRNA and protein of PPARγ, which is the key factor of adipogenesis. These cells also showed a concomitant increase in ligand-dependent activation of PPARγ target genes compared with wild-type iMEFs. Moreover, Bach1-deficient iMEFs efficiently differentiated to adipocyte compared with wild-type cells in the presence of PPARγ ligands. Our results suggest that BACH1 regulates expression of adipocyte-related genes including Pparg and potentiates adipocyte differentiation capacity. PMID:27030212

  2. Novel Genes Implicated in Embryonal, Alveolar, and Pleomorphic Rhabdomyosarcoma: A Cytogenetic and Molecular Analysis of Primary Tumors1

    PubMed Central

    Goldstein, Myriam; Meller, Isaac; Issakov, Josephine; Orr-Urtreger, Avi

    2006-01-01

    Abstract Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, likely results from deregulation of the skeletal myogenesis program. Although associations between PAX3, PAX7, FOXO1A, and RMS tumorigenesis are well recognized, the entire spectrum of genetic factors underlying RMS development and progression is unclear. Using a combined approach of spectral karyotyping, array-based comparative genomic hybridization (CGH), and expression analysis, we examined 10 primary RMS tumors, including embryonal, alveolar, and the rare adult pleomorphic variant, to explore the involvement of different genes and genetic pathways in RMS tumorigenesis. A complete karyotype established for each tumor revealed a high aneuploidy level, mostly tetraploidy, with double minutes and additional structural aberrations. Quantitative expression analysis detected the overexpression of the AURKA gene in all tumors tested, suggesting a role for this mitotic regulator in the aneuploidy and chromosomal instability observed in RMS. Array-based CGH analysis in primary RMS tumors detected copy number changes of genes involved in multiple genetic pathways, including transcription factors such as MYC-related gene from lung cancer and the cytoskeleton and cell adhesion-encoding genes laminin γ-2 and p21-activated kinase-1. Our data suggest the involvement of genes encoding cell adhesion, cytoskeletal signaling, and transcriptional and cell cycle components in RMS tumorigenesis. PMID:16790082

  3. Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells

    SciTech Connect

    Mendez, M.J.; Abderrahim, H.; Noguchi, M.

    1995-03-20

    With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (V{sub H}) genes, the major diversity (D) gene cluster, the joining (J{sub H}) genes, the intronic enhancer (E{sub H}), and the constant region genes, mu (C{mu}) and delta (C{delta}). Two IgK locus-derived YACs each contain three variable (V{kappa}) genes, the joining (J{kappa}) region, the intronic enhancer (E{kappa}), the constant gene (C{kappa}), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form. 78 refs., 7 figs.

  4. Generation of KCL029 research grade human embryonic stem cell line carrying a mutation in WAS gene.

    PubMed

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL029 human embryonic stem cell line was derived from an embryo donated for research that carried a c.814T>C mutation in the WAS gene, which is linked to the Wiskott-Aldrich syndrome, a rare, inherited, X-linked, recessive disease characterized by immune dysregulation and microthrombocytopenia. The line is also carrier for a mutation p.N1152H in the gene encoding the cystic fibrosis transmembrane conductance regulator CFTR. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345811

  5. Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

    PubMed Central

    Katsman, Diana; Stackpole, Emma J.; Domin, Daniel R.; Farber, Debora B.

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation. PMID:23226281

  6. Gene Expression Profiling of Shoot-Derived Calli from Adult Radiata Pine and Zygotic Embryo-Derived Embryonal Masses

    PubMed Central

    Garcia-Mendiguren, O.; Montalbán, I. A.; Stewart, D.; Moncaleán, P.; Klimaszewska, K.; Rutledge, R. G.

    2015-01-01

    Background Although somatic embryogenesis has an unprecedented potential for large-scale clonal propagation of conifers, the ability to efficiently induce the embryonal cultures required for somatic embryo production has long been a challenge. Furthermore, because early stage zygotic embryos remain the only responsive explants for pines, it is not possible to clone individual trees from vegetative explants at a commercial scale. This is of particular interest for adult trees because many elite characteristics only become apparent following sexual maturation. Findings Shoot explants collected from adult radiata pine trees were cultured on four induction media differing in plant growth regulator composition, either directly after collection or from in vitro-generated axillary shoots. Six callus lines were selected for microscopic examination, which failed to reveal any embryonal masses (EM). qPCR expression profiling of five of these lines indicated that explant type influenced the absolute level of gene expression, but not the type of genes that were expressed. The analysis, which also included three EM lines induced from immature zygotic embryos, encompassed five categories of genes reflective of metabolic, mitotic and meristematic activity, along with putative markers of embryogenicity. Culture medium was found to have no significant impact on gene expression, although differences specific to the explant’s origin were apparent. Expression of transcriptional factors associated with vegetative meristems further suggested that all of the callus lines possessed a substantive vegetative character. Most notable, however, was that they all also expressed a putative embryogenic marker (LEC1). Conclusions While limited in scope, these results illustrate the utility of expression profiling for characterizing tissues in culture. For example, although the biological significance of LEC1 expression is unclear, it does present the possibility that these callus lines possess

  7. The Homeotic Gene Sex Combs Reduced of Drosophila Melanogaster Is Differentially Regulated in the Embryonic and Imaginal Stages of Development

    PubMed Central

    Pattatucci, A. M.; Kaufman, T. C.

    1991-01-01

    The Sex combs reduced (Scr) locus is unique among the genes contained within the Antennapedia complex (ANT-C) of Drosophila melanogaster in that it directs functions that are required for both cephalic and thoracic development in the embryo and the adult. Antibodies raised against protein encoded by Scr were used to follow the distribution of this gene product in embryos and imaginal discs of third instar larvae. Analysis of Scr protein accumulation in embryos hemizygous for breakpoint lesions mapping throughout the locus has allowed us to determine that sequences required for establishment of the Scr embryonic pattern are contained within a region of DNA that overlaps with the identified upstream regulatory region of the segmentation gene fushi tarazu (ftz). Gain-of-function mutations in Scr result in the presence of ectopic sex comb teeth on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Heterozygous combinations of gain-of-function alleles with a wild-type Scr gene exhibit no evidence of ectopic protein localization in the second and third thoracic segments of embryos. However, mesothoracic and metathoracic leg imaginal discs can be shown to accumulate ectopically expressed Scr protein, implying a differential regulation of the Scr gene during these two periods of development. Additionally, we have found that the spatial pattern of Scr gene expression in imaginal tissues involved in the development of the adult thorax is governed in part by synapsis of homologous chromosomes in this region of the ANT-C. However, those imaginal discs that arise anteriorly to the prothorax do not appear to be sensitive to this form of gene regulation. Finally, we have demonstrated that the extent of Scr expression is influenced by mutations at the Polycomb (Pc) locus but not by mutant alleles of the zeste (z) gene. Taken together, our data suggests that Scr gene expression is differentially regulated both temporally and spatially in a manner that is

  8. Hemoglobin Agenogi--A rare abnormal beta globin chain variant.

    PubMed

    Sharma, Sunita; Sharma, Geetika; Chandra, Jagdish; Colah, Roshan

    2016-01-01

    Haemoglobin (Hb) Agenogi is clinically asymptomatic, rare β-globin chain variant characterized by a substitution of glutamic acid by lysine at position 90 of β-chain. It elutes in the C-window on high-performance liquid chromatography (HPLC). We report a 10-year-old male with easy fatigability, lethargy, pallor, and mild splenomegaly. Hematological parameters revealed microcytic hypochromic anemia and mildly raised red blood cells count, suggestive of thalassemia trait. On HPLC, a predominant peak was observed in the C-window (82.6%) along with raised HbA 2 level (9.3%). Based on these findings, a possibility of HbC disease/β-thalassemia trait doubly heterozygous was considered. Family studies were advised. HPLC findings in father were suggestive of β-thalassemia trait, while both his mother and brother had an abnormal peak in the C-window of 42.7% and 40.8%, respectively, with elevated HbA 2 values of 5% and 4.9%, respectively. Direct DNA sequencing revealed intervening sequences 1-5 (G ; C) in father, confirming β-thalassemia trait. His mother and brother had heterozygous gene mutation at codon 90 of β-globin chain (G ; A) suggestive of Hb Agenogi. The child carried mutations for both β-thalassemia trait as well as Hb Agenogi. PMID:26960650

  9. PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.

    PubMed

    Teo, Adrian Kee Keong; Tsuneyoshi, Norihiro; Hoon, Shawn; Tan, Ee Kim; Stanton, Lawrence W; Wright, Christopher V E; Dunn, N Ray

    2015-04-14

    Inactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis, which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However, little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1, whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type. PMID:25843046

  10. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    PubMed Central

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  11. Eye-Specific Gene Expression following Embryonic Ethanol Exposure in Zebrafish: Roles for Heat Shock Factor 1

    PubMed Central

    Kashyap, Bhavani; Pegorsch, Laurel; Frey, Ruth A.; Sun, Chi; Shelden, Eric A.; Stenkamp, Deborah L.

    2014-01-01

    The mechanisms through which ethanol exposure results in developmental defects remain unclear. We used the zebrafish model to elucidate eye-specific mechanisms that underlie ethanol-mediated microphthalmia (reduced eye size), through time-series microarray analysis of gene expression within eyes of embryos exposed to 1.5% ethanol. 62 genes were differentially expressed (DE) in ethanol-treated as compared to control eyes sampled during retinal neurogenesis (24-48 hours post-fertilization). The EDGE (extraction of differential gene expression) algorithm identified >3000 genes DE over developmental time in ethanol-exposed eyes as compared to controls. The DE lists included several genes indicating a mis-regulated cellular stress response due to ethanol exposure. Combined treatment with sub-threshold levels of ethanol and a morpholino targeting heat shock factor 1 mRNA resulted in microphthalmia, suggesting convergent molecular pathways. Thermal preconditioning partially prevented ethanol-mediated microphthalmia while maintaining Hsf-1 expression. These data suggest roles for reduced Hsf-1 in mediating microphthalmic effects of embryonic ethanol exposure. PMID:24355176

  12. Gene expression classification using epigenetic features and DNA sequence composition in the human embryonic stem cell line H1.

    PubMed

    Su, Wen-Xia; Li, Qian-Zhong; Zhang, Lu-Qiang; Fan, Guo-Liang; Wu, Cheng-Yan; Yan, Zhen-He; Zuo, Yong-Chun

    2016-10-30

    Epigenetic factors are known to correlate with gene expression in the existing studies. However, quantitative models that accurately classify the highly and lowly expressed genes based on epigenetic factors are currently lacking. In this study, a new machine learning method combines histone modifications, DNA methylation, DNA accessibility, transcription factors, and trinucleotide composition with support vector machines (SVM) is developed in the context of human embryonic stem cell line (H1). The results indicate that the predictive accuracy will be markedly improved when the epigenetic features are considered. The predictive accuracy and Matthews correlation coefficient of the best model are as high as 95.96% and 0.92 for 10-fold cross-validation test, and 95.58% and 0.92 for independent dataset test, respectively. Our model provides a good way to judge a gene is either highly or lowly expressed gene by using genetic and epigenetic data, when the expression data of the gene is lacking. And a web-server GECES for our analysis method is established at http://202.207.14.87:8032/fuwu/GECES/index.asp, so that other scientists can easily get their desired results by our web-server, without going through the mathematical details. PMID:27468948

  13. P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Mutations Affecting Embryonic Pns Development

    PubMed Central

    Salzberg, A.; Prokopenko, S. N.; He, Y.; Tsai, P.; Pal, M.; Maroy, P.; Glover, D. M.; Deak, P.; Bellen, H. J.

    1997-01-01

    To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P-element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens. PMID:9409832

  14. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

    PubMed

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  15. Selection for divergent body size alters rates of embryonic skeletal muscle formation and muscle gene expression patterns.

    PubMed

    Lu, Yue; Bradley, Jennifer S; Siegel, Paul B; Yang, Ning; Johnson, Sally E

    2015-12-01

    The impact of divergent selection for body size on embryogenesis is poorly understood. The objective of this experiment was to document skeletal muscle development during embryogenesis in two lines of chickens that display divergent growth as adults. Results reveal that after 54 generations of opposing selection from a common founder population, the embryos from the low weight select (LWS) line develop more rapidly during early embryogenesis than those from the high weight select (HWS) line. Muscle formation during the late embryonic period is more rapid and extensive in the HWS embryo than in the LWS contemporary. Isolated muscle progenitors from embryonic day 10 HWS embryos proliferated more rapidly, forming fibers sooner with a larger size than the LWS cells. The limited myogenic capacity of the LWS progenitor cells is not attributed to altered patterns of expression of Pax7, Pax3 or the myogenic regulatory factor genes. Members of the fibroblast growth factor family are potent mitogens and inhibitors of myoblast differentiation. Transcript abundance of FGF2 and FGF4 was measured in cultures of HWS and LWS progenitors as a function of time. The pattern of expression of FGF4 was similar between HWS and LWS with a large increase between days 1 and 3 followed by a reduction at day 5 of culture. Expression of FGF2 in LWS muscle cells did not change while a significant reduction in FGF2 expression was observed by day 5 in the HWS. Our results indicate that divergent selection for postnatal growth has altered embryonic development. PMID:26660844

  16. Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene

    PubMed Central

    Yong, Carmen S. M.; Sharkey, Janelle; Duscio, Belinda; Venville, Ben; Wei, Wei-Zen; Jones, Richard F.; Slaney, Clare Y.; Mir Arnau, Gisela; Papenfuss, Anthony T.

    2015-01-01

    The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes. PMID:26334628

  17. An Expressed Retrogene of the Master Embryonic Stem Cell Gene POU5F1 Is Associated with Prostate Cancer Susceptibility

    PubMed Central

    Breyer, Joan P.; Dorset, Daniel C.; Clark, Travis A.; Bradley, Kevin M.; Wahlfors, Tiina A.; McReynolds, Kate M.; Maynard, William H.; Chang, Sam S.; Cookson, Michael S.; Smith, Joseph A.; Schleutker, Johanna; Dupont, William D.; Smith, Jeffrey R.

    2014-01-01

    Genetic association studies of prostate and other cancers have identified a major risk locus at chromosome 8q24. Several independent risk variants at this locus alter transcriptional regulatory elements, but an affected gene and mechanism for cancer predisposition have remained elusive. The retrogene POU5F1B within the locus has a preserved open reading frame encoding a homolog of the master embryonic stem cell transcription factor Oct4. We find that 8q24 risk alleles are expression quantitative trait loci correlated with reduced expression of POU5F1B in prostate tissue and that predicted deleterious POU5F1B missense variants are also associated with risk of transformation. POU5F1 is known to be self-regulated by the encoded Oct4 transcription factor. We further observe that POU5F1 expression is directly correlated with POU5F1B expression. Our results suggest that a pathway critical to self-renewal of embryonic stem cells may also have a role in the origin of cancer. PMID:24581739

  18. Gene-environment interaction demonstrates the vulnerability of the embryonic heart.

    PubMed

    O'Reilly, Victoria C; Lopes Floro, Kylie; Shi, Hongjun; Chapman, Bogdan E; Preis, Jost I; James, Alexander C; Chapman, Gavin; Harvey, Richard P; Johnson, Randall S; Grieve, Stuart M; Sparrow, Duncan B; Dunwoodie, Sally L

    2014-07-01

    Mammalian embryos develop in a low oxygen environment. The transcription factor hypoxia inducible factor 1a (HIF1α) is a key element in the cellular response to hypoxia. Complete deletion of Hif1α from the mouse conceptus causes extensive placental, vascular and heart defects, resulting in embryonic lethality. However the precise role of Hif1α in each of these organ systems remains unknown. To further investigate, we conditionally-deleted Hif1α from mesoderm, vasculature and heart individually. Surprisingly, deletion from these tissues did not recapitulate the same severe heart phenotype or embryonic lethality. Placental insufficiency, such as occurs in the complete Hif1α null, results in elevated cellular hypoxia in mouse embryos. We hypothesized that subjecting the Hif1α conditional null embryos to increased hypoxic stress might exacerbate the effects of tissue-specific Hif1α deletion. We tested this hypothesis using a model system mimicking placental insufficiency. We found that the majority of embryos lacking Hif1α in the heart died when exposed to non-physiological hypoxia. This was a heart-specific phenomenon, as HIF1α protein accumulated predominantly in the myocardium of hypoxia-stressed embryos. Our study demonstrates the vulnerability of the heart to lowered oxygen levels, and that under such conditions of non-physiological hypoxia the embryo absolutely requires Hif1α to continue normal development. Importantly, these findings extend our understanding of the roles of Hif1α in cardiovascular development. PMID:24657234

  19. Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus.

    PubMed

    Cha, Jeeyeon; Burnum-Johnson, Kristin E; Bartos, Amanda; Li, Yingju; Baker, Erin S; Tilton, Susan C; Webb-Robertson, Bobbie-Jo M; Piehowski, Paul D; Monroe, Matthew E; Jegga, Anil G; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K

    2015-06-12

    Embryonic diapause is a reproductive strategy widespread in the animal kingdom. This phenomenon is defined by a temporary arrest in blastocyst growth and metabolic activity within a quiescent uterus without implantation until the environmental and maternal milieu become favorable for pregnancy to progress. We found that uterine Msx expression persists during diapause across species; their inactivation in the mouse uterus results in termination of diapause with the development of implantation-like responses ("pseudoimplantation") that ultimately succumbed to resorption. To understand the cause of this failure, we compared proteome profiles between floxed and Msx-deleted uteri. In deleted uteri, several functional networks, including transcription/translation, ubiquitin-proteasome, inflammation, and endoplasmic reticulum stress, were dysregulated. Computational modeling predicted intersection of these pathways on an enhanced inflammatory signature. Further studies showed that this signature was reflected in increased phosphorylated IκB levels and nuclear NFκB in deleted uteri. This was associated with enhanced proteasome activity and endoplasmic reticulum stress. Interestingly, treatment with anti-inflammatory glucocorticoid (dexamethasone) reduced the inflammatory signature with improvement of the diapause phenotype. These findings highlight an unexpected role of uterine Msx in limiting aberrant inflammatory responses to maintain embryonic diapause. PMID:25931120

  20. Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus*

    PubMed Central

    Cha, Jeeyeon; Burnum-Johnson, Kristin E.; Bartos, Amanda; Li, Yingju; Baker, Erin S.; Tilton, Susan C.; Webb-Robertson, Bobbie-Jo M.; Piehowski, Paul D.; Monroe, Matthew E.; Jegga, Anil G.; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K.

    2015-01-01

    Embryonic diapause is a reproductive strategy widespread in the animal kingdom. This phenomenon is defined by a temporary arrest in blastocyst growth and metabolic activity within a quiescent uterus without implantation until the environmental and maternal milieu become favorable for pregnancy to progress. We found that uterine Msx expression persists during diapause across species; their inactivation in the mouse uterus results in termination of diapause with the development of implantation-like responses (“pseudoimplantation”) that ultimately succumbed to resorption. To understand the cause of this failure, we compared proteome profiles between floxed and Msx-deleted uteri. In deleted uteri, several functional networks, including transcription/translation, ubiquitin-proteasome, inflammation, and endoplasmic reticulum stress, were dysregulated. Computational modeling predicted intersection of these pathways on an enhanced inflammatory signature. Further studies showed that this signature was reflected in increased phosphorylated IκB levels and nuclear NFκB in deleted uteri. This was associated with enhanced proteasome activity and endoplasmic reticulum stress. Interestingly, treatment with anti-inflammatory glucocorticoid (dexamethasone) reduced the inflammatory signature with improvement of the diapause phenotype. These findings highlight an unexpected role of uterine Msx in limiting aberrant inflammatory responses to maintain embryonic diapause. PMID:25931120

  1. Cloning, characterization, and embryonic expression analysis of the Drosophila melanogaster gene encoding insulin/relaxin-like peptide.

    PubMed

    Nasonkin, Igor O; Alikasifoglu, Ayfer; Barrette, Terry; Cheng, Michael M; Thomas, Pamela M; Nikitin, Alexey G

    2002-07-12

    Insulin is one of the key peptide hormones that regulates growth and metabolism in vertebrates. Evolutionary conservation of many elements of the insulin/IGF signaling network makes it possible to study the basic genetic function of this pathway in lower metazoan models such as Drosophila. Here we report the cloning and characterization of the gene for Drosophila insulin/relaxin-like peptide (DIRLP). The predicted protein structure of DIRLP greatly resembles typical insulin structure and contains features that differentiate it from the Drosophila juvenile hormone, another member of the insulin family. The Dirlp gene is represented as a single copy in the Drosophila melanogaster genome (compared to multiple copies for Drosophila juvenile hormone) and shows evolutionary conservation of genetic structure. The gene was mapped to the Drosophila chromosome 3, region 67D2. In situ hybridization of whole-mount Drosophila embryos with Dirlp antisense RNA probe reveals early embryonic mesodermal/ventral furrow expression pattern, consistent with earlier observation of the insulin protein immunoreactivity in Drosophila embryos. The in situ hybridization pattern was found to be identical to that obtained during immunohistochemistry analysis of the Drosophila embryos using various insulin monoclonal and polyclonal antibodies that do not recognize Drosophila juvenile hormone, supporting the idea that Dirlp is a possible Drosophila insulin ortholog. Identification of the gene for DIRLP provides a new approach for study of the regulatory pathway of the insulin family of peptides. PMID:12150949

  2. Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

    PubMed Central

    Reid, L H; Gregg, R G; Smithies, O; Koller, B H

    1990-01-01

    We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene. PMID:2349238

  3. Proteomic Shifts in Embryonic Stem Cells with Gene Dose Modifications Suggest the Presence of Balancer Proteins in Protein Regulatory Networks

    PubMed Central

    Mao, Lei; Zabel, Claus; Herrmann, Marion; Nolden, Tobias; Mertes, Florian; Magnol, Laetitia; Chabert, Caroline; Hartl, Daniela; Herault, Yann; Delabar, Jean Maurice; Manke, Thomas; Himmelbauer, Heinz; Klose, Joachim

    2007-01-01

    Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of “balancer” proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the “elasticity” of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions. PMID:18043732

  4. Embryonic develop-associated gene 1 is overexpressed and acts as a tumor promoter in thyroid carcinoma.

    PubMed

    Xu, Jinkai; Li, Zongyu; Su, Qinghua; Zhao, Jun; Ma, Jiancang

    2016-07-01

    Embryonic develop-associated gene 1 (EDAG-1), a hematopoietic tissue-specific protein, is usually highly expressed in the placenta, fetal liver, bone marrow and leukemia cells, but the expression status in normal or solid tumor tissues is rarely reported. In this study, we found that EDAG-1 was up-regulated in thyroid carcinoma tissues and cells. Knockdown of EDAG-1 suppressed proliferation and enhanced cisplatin-induced apoptosis of thyroid carcinoma cells. We also demonstrated that knockdown of EDAG-1 inactivated the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway in vitro and in vivo. Moreover, knockdown of EDAG-1 suppressed tumorigenesis of thyroid carcinoma in vivo. Taken together, these results suggest that EDAG-1 regulates the proliferation and apoptosis of thyroid carcinoma via the PI3K/Akt signaling pathway. PMID:27261581

  5. Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.

    PubMed

    Di Micco, Raffaella; Fontanals-Cirera, Barbara; Low, Vivien; Ntziachristos, Panagiotis; Yuen, Stephanie K; Lovell, Claudia D; Dolgalev, Igor; Yonekubo, Yoshiya; Zhang, Guangtao; Rusinova, Elena; Gerona-Navarro, Guillermo; Cañamero, Marta; Ohlmeyer, Michael; Aifantis, Iannis; Zhou, Ming-Ming; Tsirigos, Aristotelis; Hernando, Eva

    2014-10-01

    Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation. PMID:25263550

  6. O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter.

    PubMed

    Zhang, Zhen; Costa, Flávia C; Tan, Ee Phie; Bushue, Nathan; DiTacchio, Luciano; Costello, Catherine E; McComb, Mark E; Whelan, Stephen A; Peterson, Kenneth R; Slawson, Chad

    2016-07-22

    One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter. PMID:27231347

  7. Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.

    PubMed

    Yeo, Seungeun; Jeong, Sangkyun; Kim, Janghwan; Han, Jee-Soo; Han, Yong-Mahn; Kang, Yong-Kook

    2007-08-01

    Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation. PMID:17548060

  8. Parental RNA interference of genes involved in embryonic development of the western corn rootworm, Diabrotica virgifera virgifera LeConte.

    PubMed

    Khajuria, Chitvan; Vélez, Ana M; Rangasamy, Murugesan; Wang, Haichuan; Fishilevich, Elane; Frey, Meghan L F; Carneiro, Newton Portilho; Gandra, Premchand; Narva, Kenneth E; Siegfried, Blair D

    2015-08-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management and one of the most likely target pest species for transgenic plants that express double stranded RNA (dsRNA) is the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm cause lethality in the larval stage. In this study, we describe RNAi-mediated knockdown of two developmental genes, hunchback (hb) and brahma (brm), in the western corn rootworm delivered via dsRNA fed to adult females. dsRNA feeding caused a significant decrease in hb and brm transcripts in the adult females. Although total oviposition was not significantly affected, there was almost complete absence of hatching in the eggs collected from females exposed to dsRNA for either gene. These results confirm that RNAi is systemic in nature for western corn rootworms. These results also indicate that hunchback and brahma play important roles in rootworm embryonic development and could provide useful RNAi targets in adult rootworms to prevent crop injury by impacting the population of larval progeny of exposed adults. The ability to deliver dsRNA in a trans-generational manner by feeding to adult rootworms may offer an additional approach to utilizing RNAi for rootworm pest management. The potential to develop parental RNAi technology targeting progeny of adult rootworms in combination with Bt proteins or dsRNA lethal to larvae may increase opportunities to develop sustainable approaches to rootworm management involving RNAi technologies for rootworm control. PMID:26005118

  9. Neuron-like differentiation and selective ablation of undifferentiated embryonic stem cells containing suicide gene with Oct-4 promoter.

    PubMed

    Hara, Akira; Aoki, Hitomi; Taguchi, Ayako; Niwa, Masayuki; Yamada, Yasuhiro; Kunisada, Takahiro; Mori, Hideki

    2008-08-01

    In vivo transplantation of undifferentiated embryonic stem (ES) cells can produce teratomas with uncontrolled cell proliferation. Although ES cells may be attractive candidates for human cell-replacement therapy in the future, the major limitation of its application to the therapy is teratoma formation. In the present study, ES cells containing herpes simplex virus-thymidine kinase (HSV-tk) transgene for a suicide gene expression under the control of the Oct-4 promoter was used for ablation of undifferentiated ES cells, which may produce teratomas, using three-dimensional cell culture system allowing a multilayer cell construct. Selective ablation of undifferentiated ES cells expressing HSV-tk gene under the control of Oct-4 promoter was achieved by ganciclovir treatment. Surviving ES cells after ganciclovir treatment expressed several neuron-associated markers such as synaptophysin, beta-tubulin, vesicular glutamate transporter 1, syntaxin, protein kinase C and glial fibrillary acidic protein (GFAP) but not Oct-4. Coexpression of synaptophysin as a marker of neuronal synapse and GFAP as that of glial fibers in the surviving ES cells revealed finely structured neuronal network. Furthermore, decrease of Ki-67 proliferative index was detected in the surviving ES cells. In conclusion, selective ablation of undifferentiated ES cells by a suicide gene decreases proliferative activity and induces neuron-like differentiation in ES cells. PMID:18393636

  10. CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.

    PubMed

    Kalebic, Nereo; Taverna, Elena; Tavano, Stefania; Wong, Fong Kuan; Suchold, Dana; Winkler, Sylke; Huttner, Wieland B; Sarov, Mihail

    2016-03-01

    We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development. PMID:26758805

  11. Dual mode of embryonic development is highlighted by expression and function of Nasonia pair-rule genes

    PubMed Central

    Rosenberg, Miriam I; Brent, Ava E; Payre, François; Desplan, Claude

    2014-01-01

    Embryonic anterior–posterior patterning is well understood in Drosophila, which uses ‘long germ’ embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use ‘short germ’ embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp Nasonia (Nv) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in Nasonia suggest long germ embryogenesis. However, the Nasonia pair-rule genes even-skipped, odd-skipped, runt and hairy are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of Nv eve, odd or h causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that Nasonia uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling Drosophila at the anterior and ancestral Tribolium at the posterior. DOI: http://dx.doi.org/10.7554/eLife.01440.001 PMID:24599282

  12. Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

    SciTech Connect

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. )

    1994-05-01

    The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

  13. Spectrum of Common α-Globin Deletion Mutations in the Southern Region of Vietnam.

    PubMed

    Bui Thi Kim, Ly; Phu Chi, Dung; Hoang Thanh, Chi

    2016-06-01

    The common deletion mutations of α-globin genes in the Vietnamese population is not well known. Here we report the presence of five deletional mutations of Southeast Asia in the southern region of Vietnam. The - -(SEA) (NG_000006.1: g.26264_45564del19301) mutation is the most common type of deletion (87.35%), followed by the -α(3.7) (rightward) (NG_000006.1: g.34164_37967del3804) deletion (9.64%), -α(4.2) (leftward) (AF221717) deletion (2.41%) and - -(THAI) (NG_000006.1: g.10664_44164del33501) (0.6%) mutation in this region. The - -(FIL) (NG_000006.1: g.11684_43534del31581) mutation was not detected in this study. This result provided a view of the distribution of common α-globin gene mutations in Vietnam and could serve as a baseline for further investigations into these genetic defects. PMID:27117571

  14. Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

    PubMed Central

    Salvatori, Francesca; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Brognara, Eleonora; Lampronti, Ilaria; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2013-01-01

    In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations. PMID:19810011

  15. Repression of apical homeobox genes is required for embryonic root development in Arabidopsis.

    PubMed

    Grigg, Stephen P; Galinha, Carla; Kornet, Noortje; Canales, Claudia; Scheres, Ben; Tsiantis, Miltos

    2009-09-15

    Development of seed plant embryos is polarized along the apical-basal axis. This polarization occurs in the absence of cell migration and culminates in the establishment of two distinct pluripotent cell populations: the shoot apical meristem (SAM) and root meristem (RM), which postembryonically give rise to the entire shoot and root systems of the plant. The acquisition of genetic pathways that delimit root from shoot during embryogenesis must have played a pivotal role during land plant evolution because roots evolved after shoots in ancestral vascular plants and may be shoot-derived organs. However, such pathways are very poorly understood. Here we show that RM establishment in the model plant Arabidopsis thaliana requires apical confinement of the Class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) proteins PHABULOSA (PHB) and PHAVOLUTA (PHV), which direct both SAM development and shoot lateral organ polarity. Failure to restrict PHB and PHV expression apically via a microRNA-dependent pathway prevents correct elaboration of the embryonic root development program and results in embryo lethality. As such, repression of a fundamental shoot development pathway is essential for correct root development. Additionally, our data suggest that a single patterning process, based on HD-ZIP III repression, mediates both apical-basal and radial polarity in the embryo and lateral organ polarity in the shoot. PMID:19646874

  16. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    PubMed Central

    2011-01-01

    Background Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs). Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1. To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb

  17. The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

    PubMed Central

    Bader, Joel S.; Friedmann, Theodore

    2013-01-01

    The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease. PMID:24130677

  18. c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells

    PubMed Central

    Nie, Zuqin; Hu, Gangqing; Wei, Gang; Cui, Kairong; Yamane, Arito; Resch, Wolfgang; Wang, Ruoning; Green, Douglas R.; Tessarollo, Lino; Casellas, Rafael; Zhao, Keji; Levens, David

    2012-01-01

    Summary The c-Myc HLH-bZIP protein has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism and differentiation at the cellular, tissue or organismal levels via regulation of numerous target genes. No principle yet unifies Myc action due partly to an incomplete inventory and functional accounting of Myc’s targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II and chromatin modifications were compared during lymphocyte activation and in ES cells as well. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a non-linear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action explains the vast majority of Myc biology observed in literature. PMID:23021216

  19. Development of retrovirus vectors useful for expressing genes in cultured murine embryonal cells and hematopoietic cells in vivo.

    PubMed Central

    Guild, B C; Finer, M H; Housman, D E; Mulligan, R C

    1988-01-01

    A series of retrovirus vectors were constructed in which cellular promoter elements derived from the chicken beta-actin and human histone H4 genes were introduced within the proviral transcriptional unit of Moloney murine leukemia virus in order to promote expression of inserted sequences. Each of these vectors gave rise to high titer of virus capable of transferring the expected proviral structure to cells. Inclusion of normal 5' splice sequences or a portion of viral gag sequences in these constructions resulted in significant increases in virus titer. Each construction was transcriptionally active in NIH 3T3 cells and in undifferentiated F9 cells. One of the vectors, HSG-neo, which contained the human histone H4 promoter, was shown to be transcriptionally active in hematopoietic cells derived from long-term reconstituted bone marrow transplant recipients engrafted with transduced stem cells. These vectors should be of general use for obtaining efficient gene expression in embryonal and hematopoietic cells. Images PMID:3418785

  20. The embryonic RNA helicase gene (ERH): a new member of the DEAD box family of RNA helicases.

    PubMed Central

    Sowden, J; Putt, W; Morrison, K; Beddington, R; Edwards, Y

    1995-01-01

    DEAD box proteins share several highly conserved motifs including the characteristic Asp-Glu-Ala-Asp (D-E-A-D in the amino acid single-letter code) motif and have established or putative ATP-dependent RNA helicase activity. These proteins are implicated in a range of cellular processes that involve regulation of RNA function, including translation initiation, RNA splicing and ribosome assembly. Here we describe the isolation and characterization of an embryonic RNA helicase gene, ERH, which maps to mouse chromosome 1 and encodes a new member of the DEAD box family of proteins. The predicted ERH protein shows high sequence similarity to the testes-specific mouse PL10 and to the maternally acting Xenopus An3 helicase proteins. The ERH expression profile is similar, to that of An3, which localizes to the animal hemisphere of oocytes and is abundantly expressed in the embryo. ERH is expressed in oocytes and is a ubiquitous mRNA in the 9 days-post-conception embryo, and at later stages of development shows a more restricted pattern of expression in brain and kidney. The similarities in sequence and in expression profile suggest that ERH is the murine equivalent of the Xenopus An3 gene, and we propose that ERH plays a role in translational activation of mRNA in the oocyte and early embryo. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8948440

  1. Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.

    PubMed

    Cassar, Paul A; Carpenedo, Richard L; Samavarchi-Tehrani, Payman; Olsen, Jonathan B; Park, Chang Jun; Chang, Wing Y; Chen, Zhaoyi; Choey, Chandarong; Delaney, Sean; Guo, Huishan; Guo, Hongbo; Tanner, R Matthew; Perkins, Theodore J; Tenenbaum, Scott A; Emili, Andrew; Wrana, Jeffrey L; Gibbings, Derrick; Stanford, William L

    2015-10-01

    In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN. PMID:26265008

  2. Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function

    PubMed Central

    Yeung, A. T. Y.; Hale, C.; Xia, J.; Tate, P. H.; Goulding, D.; Keane, J. A.; Mukhopadhyay, S.; Forrester, L.; Billker, O.; Skarnes, W. C.; Hancock, R. E. W.; Dougan, G.

    2015-01-01

    The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection. PMID:25752829

  3. Comparative analysis of Hox paralog group 2 gene expression during Nile tilapia (Oreochromis niloticus) embryonic development.

    PubMed

    Le Pabic, Pierre; Stellwag, Edmund J; Brothers, Shelby N; Scemama, Jean-Luc

    2007-12-01

    The hindbrain and pharyngeal arch-derived structures of vertebrates are determined, at least in part, by Hox paralog group 2 genes. In sarcopterygians, the Hoxa2 gene alone appears to specify structures derived from the second pharyngeal arch (PA2), while in zebrafish (Danio rerio), either of the two Hox PG2 genes, hoxa2b or hoxb2a, can specify PA2-derived structures. We previously reported three Hox PG2 genes in striped bass (Morone saxatilis), including hoxa2a, hoxa2b, and hoxb2a and observed that only HoxA cluster genes are expressed in PA2, indicative that they function alone or together to specify PA2. In this paper, we present the cloning and expression analysis of Nile tilapia (Oreochromis niloticus) Hox PG2 genes and show that all three genes are expressed in the hindbrain and in PA2. The expression of hoxb2a in PA2 was unexpected given the close phylogenetic relationship of Nile tilapia and striped bass, both of which are members of the order Perciformes. A reanalysis of striped bass hoxb2a expression demonstrated that it is expressed in PA2 with nearly the same temporal and spatial expression pattern as its Nile tilapia ortholog. Further, we determined that Nile tilapia and striped bass hoxa2a orthologs are expressed in PA2 well beyond the onset of chondrogenesis whereas neither hoxa2b nor hoxb2a expression persist until this stage, which, according to previous hypotheses, suggests that hoxa2a orthologs in these two species function alone as selector genes of PA2 identity. PMID:17924140

  4. Embryonic Stem Cell (ES)-Specific Enhancers Specify the Expression Potential of ES Genes in Cancer.

    PubMed

    Aran, Dvir; Abu-Remaileh, Monther; Levy, Revital; Meron, Nurit; Toperoff, Gidon; Edrei, Yifat; Bergman, Yehudit; Hellman, Asaf

    2016-02-01

    Cancers often display gene expression profiles resembling those of undifferentiated cells. The mechanisms controlling these expression programs have yet to be identified. Exploring transcriptional enhancers throughout hematopoietic cell development and derived cancers, we uncovered a novel class of regulatory epigenetic mutations. These epimutations are particularly enriched in a group of enhancers, designated ES-specific enhancers (ESSEs) of the hematopoietic cell lineage. We found that hematopoietic ESSEs are prone to DNA methylation changes, indicative of their chromatin activity states. Strikingly, ESSE methylation is associated with gene transcriptional activity in cancer. Methylated ESSEs are hypermethylated in cancer relative to normal somatic cells and co-localized with silenced genes, whereas unmethylated ESSEs tend to be hypomethylated in cancer and associated with reactivated genes. Constitutive or hematopoietic stem cell-specific enhancers do not show these trends, suggesting selective reactivation of ESSEs in cancer. Further analyses of a hypomethylated ESSE downstream to the VEGFA gene revealed a novel regulatory circuit affecting VEGFA transcript levels across cancers and patients. We suggest that the discovered enhancer sites provide a framework for reactivation of ES genes in cancer. PMID:26886256

  5. Embryonic Stem Cell (ES)-Specific Enhancers Specify the Expression Potential of ES Genes in Cancer

    PubMed Central

    Levy, Revital; Meron, Nurit; Toperoff, Gidon; Edrei, Yifat; Bergman, Yehudit; Hellman, Asaf

    2016-01-01

    Cancers often display gene expression profiles resembling those of undifferentiated cells. The mechanisms controlling these expression programs have yet to be identified. Exploring transcriptional enhancers throughout hematopoietic cell development and derived cancers, we uncovered a novel class of regulatory epigenetic mutations. These epimutations are particularly enriched in a group of enhancers, designated ES-specific enhancers (ESSEs) of the hematopoietic cell lineage. We found that hematopoietic ESSEs are prone to DNA methylation changes, indicative of their chromatin activity states. Strikingly, ESSE methylation is associated with gene transcriptional activity in cancer. Methylated ESSEs are hypermethylated in cancer relative to normal somatic cells and co-localized with silenced genes, whereas unmethylated ESSEs tend to be hypomethylated in cancer and associated with reactivated genes. Constitutive or hematopoietic stem cell-specific enhancers do not show these trends, suggesting selective reactivation of ESSEs in cancer. Further analyses of a hypomethylated ESSE downstream to the VEGFA gene revealed a novel regulatory circuit affecting VEGFA transcript levels across cancers and patients. We suggest that the discovered enhancer sites provide a framework for reactivation of ES genes in cancer. PMID:26886256

  6. In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes

    PubMed Central

    Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan MW

    2006-01-01

    Background Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. Results The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. Conclusion We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they

  7. elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family.

    PubMed Central

    Spieth, J; Shim, Y H; Lea, K; Conrad, R; Blumenthal, T

    1991-01-01

    The short, asymmetrical DNA sequence to which the vertebrate GATA family of transcription factors binds is present in some Caenorhabditis elegans gene regulatory regions: it is required for activation of the vitellogenin genes and is also found just 5' of the TATA boxes of tra-2 and the msp genes. In vertebrates GATA-1 is specific to erythroid lineages, whereas GATA-2 and GATA-3 are present in multiple tissues. In an effort to identify the trans-acting factors that may recognize this sequence element in C. elegans, we used a degenerate oligonucleotide to clone a C. elegans homolog to this gene. We call this gene elt-1 (erythrocytelike transcription factor). It is single copy and specifies a 1.75-kb mRNA that is present predominantly, if not exclusively, in embryos. The region of elt-1 encoding two zinc fingers is remarkably similar to the DNA-binding domain of the vertebrate GATA-binding proteins. However, outside of the DNA-binding domains the amino acid sequences are quite divergent. Nevertheless, introns are located at identical or nearly identical positions in elt-1 and the mouse GATA-1 gene. In addition, elt-1 mRNA is trans-spliced to the 22-base untranslated leader, SL1. The DNA upstream of the elt-1 TATA box contains eight copies of the GATA recognition sequence within the first 300 bp, suggesting that elt-1 may be autogenously regulated. Our results suggest that the specialized role of GATA-1 in erythroid gene expression was derived after separation of the nematodes and the line that led to the vertebrates, since C. elegans lacks an erythroid lineage. Images PMID:1875944

  8. The function of DrPax1b gene in the embryonic development of zebrafish.

    PubMed

    Liu, Xin; Wang, Hua; Li, Guang; Huang, Hui-Zhe; Wang, Yi-Quan

    2013-01-01

    Vertebrate Pax1 gene is a member of Pax gene family and encodes a transcription factor associated with crucial roles in the development of pharyngeal pouch, scletrotome and limb bud. In zebrafish, the genome contains two Pax1 paralogs, DrPax1a and DrPax1b, which share high sequence similarity with other Pax1 genes. To elucidate the function of zebrafish DrPax1b gene, we first examined the gene expression pattern and found that it was mainly expressed in the endodermal pharyngeal pouch, caudal somites, notochord, and fin bud. Then, we performed knockdown experiments using antisense morpholino oligonucleotides, which lead to the defects in the vertebral column, tail, pharyngeal skeleton, and pectoral fin. Additionally, we also found that the mouse MmPax1 mRNA, but not the amphioxus AmphiPax1/9 mRNA, could rescue the MO-induced defects. Furthermore, sequence alignment revealed that the N-terminal region of vertebrate Pax1 and amphioxus Pax1/9 were highly conserved, whereas their C-terminal regions were relatively divergent. However, the chimeric Am(N)Dr(C)Pax1, Mm(N)Dr(C)Pax1 and Dr(N)Mm(C)Pax1 mRNA could partially rescue the defects, while the Dr(N)Am(C)Pax1 mRNA could not. In conclusion, our data demonstrate a conserved function of DrPax1b in the development of the vertebral column, pectoral fin and pharyngeal skeleton formation in zebrafish and also provide critical insight into the functional evolution of Pax1 gene by changing its C-terminal sequence. PMID:24463529

  9. Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

    PubMed

    Tapia-Limonchi, Rafael; Cahuana, Gladys M; Caballano-Infantes, Estefania; Salguero-Aranda, Carmen; Beltran-Povea, Amparo; Hitos, Ana B; Hmadcha, Abdelkrim; Martin, Franz; Soria, Bernat; Bedoya, Francisco J; Tejedo, Juan R

    2016-09-01

    Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc. PMID:26853909

  10. The interferon α-responsive gene, Ifrg15, plays vital roles during mouse early embryonic development.

    PubMed

    Yang, Ye; Wang, Jiayi; Zhao, Chun; Chen, Xiaojiao; Chen, Li; Zhang, Junqiang; Huo, Ran; Liu, Chang; Tong, Hua; Ling, Xiufeng

    2016-08-01

    The interferon alpha-responsive gene (Ifrg15) mRNA is highly expressed in various stages during preimplantation mammalian embryo development. Unfortunately, few studies have investigated the effect of Ifrg15 in this process. In mammals, the fusion of male and female pronuclei generates a diploid zygote, and is an important step for subsequent cleavage and blastocyst formation. Here, by using RNA interference, rescue experiments, immunofluorescence staining and live cell observations, we found that preimplantation embryo development was arrested at the 1-cell stage after knocking down Ifrg15 expression. This induced DNA damage and prevented the cleavage of embryos. Furthermore, the effect of Ifrg15 deficiency in arresting preimplantation embryo development produced by specific short interfering RNA microinjection was concentration-dependent. Using transcriptome expression profiles, gene ontogeny functional annotation and enrichment analysis, we gained 197 enriched pathways based on 1445 differentially expressed genes (DEGs). Of these, 12 pathways and about one third of the DEGs were involved in DNA damage, DNA repair, cell cycle, and developmental processes. Thus, the IFRG15 protein might be an important molecule for maintaining genomic integrity and stability through upregulating or downregulating a cascade of genes to permit normal preimplantation embryo development. PMID:26911731

  11. Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

    PubMed Central

    Chin, Joanna Y; Reza, Faisal; Glazer, Peter M

    2013-01-01

    Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes. PMID:23337982

  12. Why the DNA self-depurination mechanism operates in HB-β but not in β-globin paralogs HB-δ, HB-ɛ1, HB-γ1 and HB-γ2.

    PubMed

    Amosova, Olga; Alvarez-Dominguez, Juan R; Fresco, Jacques R

    2015-08-01

    The human β-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5'GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5'G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of β-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and β-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the β-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in β-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human β-globin paralogs. PMID:26042536

  13. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  14. Homologous globin cell-free transcription system with comparison of heterologous factors.

    PubMed Central

    Tolunay, H E; Yang, L; Kemper, W M; Safer, B; Anderson, W F

    1984-01-01

    Mouse erythroleukemia (MEL) cells provide a useful model system to examine the regulation of globin gene expression. MEL cells ordinarily do not express globin genes, but in the presence of inducers, such as dimethyl sulfoxide or hexamethylene bisacetamide, they mimic erythroid differentiation. We have developed a cell-free transcription system from uninduced MEL cells to determine the requirements for mRNA synthesis. The MEL system directs accurate transcription of adenovirus type 2 major late DNA and mouse betamaj-globin with an efficiency comparable to those of HeLa and KB cell extracts. Using the procedure of Matsui et al. (T. Matsui, J. Segall, P.A. Weil, and R.G. Roeder, J. Biol. Chem. 255:11992-11996, 1980), we have isolated three active fractions from both MEL and HeLa cell extracts which are required for accurate transcription and have shown that equivalent fractions from MEL and HeLa cell extracts are interchangeable. Our findings suggest that the components required for initiation of transcription are similar in different cell types, at least to the extent that they can be assayed in these in vitro systems. Images PMID:6583493

  15. GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer.

    PubMed

    Escamilla-Del-Arenal, Martín; Recillas-Targa, Félix

    2008-01-01

    Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development. PMID:17984219

  16. α-Globin as a molecular target in the treatment of β-thalassemia

    PubMed Central

    Mettananda, Sachith; Gibbons, Richard J.

    2015-01-01

    The thalassemias, together with sickle cell anemia and its variants, are the world’s most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and β-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of β-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E β-thalassemia that makes up approximately half of all patients born each year with severe β-thalassemia. PMID:25869286

  17. Genomic and gene expression responses to genotoxic stress in PAC2 zebrafish embryonic cell line.

    PubMed

    Šrut, Maja; Bourdineaud, Jean-Paul; Štambuk, Anamaria; Klobučar, Göran I V

    2015-11-01

    PAC2 cell line is, along most of the developed zebrafish cell lines, poorly characterized concerning its response to genotoxicants. To define the PAC2 cell line response to different forms of genotoxic stress, we exposed the cells to model genotoxic agents (benzo[a]pyrene, B[a]P, and ethyl methanesulfonate) and subsequently monitored DNA damage and alterations by using the battery of tests, including the Comet assay, quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism. The expression of several DNA repair (xpc, xpd, hr23b, rad51, msh2) and oxidative stress response (sod (Cu/Zn)) genes was monitored as well. To obtain an indication of the PAC2 cell line metabolizing capacity, the expression of genes belonging to cyp1, cyp2 and cyp3 families was assessed upon exposure to B[a]P. Genotoxic responses were observed in all the used methods, and quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism proved to be more sensitive by revealing DNA alterations even when the Comet assay indicated lack of significant damage. The PAC2 cell line demonstrated basal and B[a]P-induced expression of several cyp genes, suggesting its ability to metabolize indirect acting xenobiotics to a certain point. Based on these results, PAC2 cells seem to be sensitive zebrafish in vitro model in the genotoxicity assessment of the direct acting genotoxicant; however, they are less sensitive toward the indirect acting genotoxicant due to their limited metabolizing properties. PMID:25612249

  18. Mono-2-ethylhexyl phthalate disrupts neurulation and modifies the embryonic redox environment and gene expression.

    PubMed

    Sant, Karilyn E; Dolinoy, Dana C; Jilek, Joseph L; Sartor, Maureen A; Harris, Craig

    2016-08-01

    Mono-2-ethylhexl phthalate (MEHP) is the primary metabolite of di-2-ethylhexyl phthalate (DEHP), a ubiquitous contaminant in plastics. This study sought to determine how structural defects caused by MEHP in mouse whole embryo culture were related to temporal and spatial patterns of redox state and gene expression. MEHP reduced morphology scores along with increased incidence of neural tube defects. Glutathione (GSH) and cysteine (Cys) concentrations fluctuated spatially and temporally in embryo (EMB) and visceral yolk sac (VYS) across the 24h culture. Redox potentials (Eh) for GSSG/GSH were increased by MEHP in EMB (12h) but not in VYS. CySS/CyS Eh in EMB and VYS were significantly increased at 3h and 24h, respectively. Gene expression at 6h showed that MEHP induced selective alterations in EMB and VYS for oxidative phosphorylation and energy metabolism pathways. Overall, MEHP affects neurulation, alters Eh, and spatially alters the expression of metabolic genes in the early organogenesis-stage mouse conceptus. PMID:27167697

  19. Metabolic suppression during mesodermal differentiation of embryonic stem cells identified by single-cell comprehensive gene expression analysis.

    PubMed

    Zhou, Yuanshu; Fujisawa, Ikuma; Ino, Kosuke; Matsue, Tomokazu; Shiku, Hitoshi

    2015-09-01

    Flk-1 (VEGF receptor 2) is a well-defined mesodermal progenitor marker and the Flk-1-positive (Flk-1(+)) cells derived from embryonic stem cells (ESCs) have been known to generate hemangioblasts and cardiovascular progenitor cells, which are formed in the early and late stages of differentiation, respectively. In this study, we separated Flk-1(+) and Flk-1(-) cells from spontaneously differentiating embryoid bodies (EBs) of mouse ESCs. We found that cell aggregates derived from late stage Flk-1(+) cells had a relatively small size and a low oxygen consumption rate (OCR) compared with those derived from Flk-1(-) cells. Furthermore, using single-cell comprehensive gene expression analysis, we found that both Flk-1(+) and Flk-1(-) cells could be categorized into subgroups with either low or high glucose metabolic activity. We observed that metabolic suppression occurs in cells expressing an intermediate level of both Nanog and Pou5f1. Taken together, our data suggested that the temporary metabolic suppression is an intrinsic feature of mesodermal differentiation. PMID:26211925

  20. Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

    PubMed Central

    Aoshima, Keisuke; Inoue, Erina; Sawa, Hirofumi; Okada, Yuki

    2015-01-01

    Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice. PMID:25925669

  1. Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development.

    PubMed

    Aoshima, Keisuke; Inoue, Erina; Sawa, Hirofumi; Okada, Yuki

    2015-07-01

    Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice. PMID:25925669

  2. Investigating alpha-globin structural variants: a retrospective review of 135,000 Brazilian individuals

    PubMed Central

    Kimura, Elza Miyuki; Oliveira, Denise Madureira; Jorge, Susan Elisabeth; Ribeiro, Daniela Maria; Zaccariotto, Tânia Regina; Santos, Magnun Nueldo Nunes; Almeida, Vanessa; Albuquerque, Dulcinéia Martins; Costa, Fernando Ferreira; Sonati, Maria de Fátima

    2015-01-01

    Background Brazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population. Methods A total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC) and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC). Mutations were identified by molecular analyses, and the oxygen affinity, heme–heme cooperativity and Bohr effect of the variants were evaluated by functional tests. Results Four new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia. Conclusion The variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations. PMID:25818820

  3. Generation of KCL021 research grade human embryonic stem cell line carrying a ΔF508 mutation in the CFTR gene.

    PubMed

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL021 human embryonic stem cell line was derived from an embryo donated for research that carried a ΔF508 mutation affecting the CFTR gene encoding the cystic fibrosis transmembrane conductance regulator. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345808

  4. RING1B O-GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells.

    PubMed

    Maury, Julien Jean Pierre; El Farran, Chadi A; Ng, Daniel; Loh, Yuin-Han; Bi, Xuezhi; Bardor, Muriel; Choo, Andre Boon-Hwa

    2015-07-01

    O-linked-N-acetylglucosamine (O-GlcNAc) post-translationally modifies and regulates thousands of proteins involved in various cellular mechanisms. Recently, O-GlcNAc has been linked to human embryonic stem cells (hESC) differentiation, however the identity and function of O-GlcNAc proteins regulating hESC remain unknown. Here, we firstly identified O-GlcNAc modified human stem cell regulators such as hnRNP K, HP1γ, and especially RING1B/RNF2. Thereafter, we focused our work on RING1B which is the catalytic subunit of the polycomb repressive complex 1 (PRC1) a major epigenetic repressor essential for pluripotency maintenance and differentiation. By point-mutation, we show that T(250)/S(251) and S(278) RING1B residues are bearing O-GlcNAc, and that T(250)/S(251) O-GlcNAcylation decreases during differentiation. O-GlcNAc seems to regulate RING1B-DNA binding as suggested by our ChIP-sequencing results. Non-O-GlcNAcylated RING1B is found to be enriched near cell cycle genes whereas O-GlcNAcylated RING1B seems preferentially enriched near neuronal genes. Our data suggest that during hESC differentiation, the decrease of RING1B O-GlcNAcylation might enable PRC1 to switch its target to induce neuron differentiation. Overall, we demonstrate that O-GlcNAc modifies and regulates an essential epigenetic tool, RING1B, which may contribute to hESC pluripotency maintenance and differentiation. PMID:26100231

  5. Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca(2+) release channel.

    PubMed

    Filipova, Dilyana; Walter, Anna M; Gaspar, John A; Brunn, Anna; Linde, Nina F; Ardestani, Mostafa A; Deckert, Martina; Hescheler, Jürgen; Pfitzer, Gabriele; Sachinidis, Agapios; Papadopoulos, Symeon

    2016-01-01

    In mature skeletal muscle, the intracellular Ca(2+) concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca(2+) release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1's potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca(2+) signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca(2+) signaling during muscle organ development. PMID:26831464

  6. Abscisic acid regulation of DC8, a carrot embryonic gene. [Daucus carota

    SciTech Connect

    Hatzopoulos, P.; Fong, F.; Sung, Z.R. Texas A M Univ., College Station )

    1990-10-01

    DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage embryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 {times} 10{sup {minus}7} molar ABA while 10{sup {minus}6} molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.

  7. Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca2+ release channel

    PubMed Central

    Filipova, Dilyana; Walter, Anna M.; Gaspar, John A.; Brunn, Anna; Linde, Nina F.; Ardestani, Mostafa A.; Deckert, Martina; Hescheler, Jürgen; Pfitzer, Gabriele; Sachinidis, Agapios; Papadopoulos, Symeon

    2016-01-01

    In mature skeletal muscle, the intracellular Ca2+ concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca2+ release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1’s potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca2+ signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca2+ signaling during muscle organ development. PMID:26831464

  8. ECM and FGF-dependent assay of embryonic submandibular gland epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during development

    PubMed Central

    Rebustini, Ivan T.; Hoffman, Matthew P.

    2012-01-01

    Epithelial-mesenchyme interactions during organogenesis are regulated by dynamic and reciprocal interactions between growth factors and extracellular matrix (ECM) components. Mouse embryonic submandibular gland (SMG) epithelium, isolated from its endogenous mesenchyme, undergoes branching morphogenesis when cultured ex vivo in a basement membrane extract in serum-free medium with growth factor stimulation. The resulting three-dimensional epithelial morphogenesis in the defined culture system makes this a useful model to analyze cell-cell and cell-matrix interactions, growth factor-mediated signaling and gene expression, proliferation, apoptosis, migration, lumen formation, and epithelial morphogenesis in a primary organ culture system. SMG epithelial culture is robust, reproducible, uses small amounts of reagents, and changes in gene expression are measured by real-time PCR using a limited amount of embryonic tissue. In this chapter, we describe a detailed protocol for isolating primary embryonic SMG epithelium and setting up an ECM and growth factor-dependent, serum-free assay of epithelial morphogenesis, with subsequent analysis of gene expression by real-time PCR. PMID:19247608

  9. A five prime splice-region G yields C mutation in exon 1 of the human. beta. -globin gene inhibits pre-mRNA splicing: A mechanism for. beta. sup + -thalassemia

    SciTech Connect

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M. ); Gattoni, R.; Stevenin, J. ); Chibani, J. )

    1989-02-01

    The authors have characterized a Mediterranean {beta}-thalassemia allele containing a sequence change at codon 30 that alters both {beta}-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G {yields} C transversion at position {minus}1 of intron 1 reduces severely the utilization of the normal 5{prime} splice site since the level of the Arg {yields} Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position {minus}1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5{prime} splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.

  10. Determination of Ligand Pathways in Globins

    PubMed Central

    Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen W.; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.

    2012-01-01

    Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  11. Clock genes, melanopsins, melatonin, and dopamine key enzymes and their modulation by light and glutamate in chicken embryonic retinal cells.

    PubMed

    Lima, Leonardo Henrique Ribeiro Graciani de; Santos, Kátia Pereira dos; Lauro Castrucci, Ana Maria de

    2011-03-01

    The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 µM glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 µM glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. PMID:21231870

  12. Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles

    PubMed Central

    2012-01-01

    Background Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5) collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon) which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM) from ambient air particles, human embryonic lung fibroblasts (HEL12469) were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls. Method For this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 μg EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina). The expression of selected genes was verified by quantitative real-time PCR. Results Dose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goeman's global test, varied, depending on the locality, between 12 to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair), the Wnt and TGF-β signaling pathways (associated

  13. Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells

    PubMed Central

    Park, Joonghoon; Park, Jong Woo; Oh, Hawmok; Maria, Fernanda S.; Kang, Jaeku; Tian, Xiuchun

    2016-01-01

    Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS) are important signaling molecules for embryonic stem cell (ESC) differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG). Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME) from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7th and 9th base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-)2ME than (+)2ME group (p < 0.05). This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05) and increased formation of beating embryoid bodies (EBs; p < 0.001). Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-)2ME (p < 0.01). Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative stressed

  14. Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation.

    PubMed

    Kapelushnik, J; Or, R; Filon, D; Nagler, A; Cividalli, G; Aker, M; Naparstek, E; Slavin, S; Oppenheim, A

    1995-10-15

    Beta-thalassemia major (TM) is caused by any of approximately 150 mutations within the beta-globin gene. To establish the degree of chimerism after bone marrow transplantation (BMT), we have performed molecular analysis of beta-globin mutations in 14 patients with TM over a period of 10 years. All patients underwent T cell-depleted allogeneic BMT from HLA-identical related donors, using either in vitro T-cell depletion with CAMPATH 1M and complement or in vivo depletion using CAMPATH 1G in the bone marrow collection bag. To date, at different time periods after BMT, seven patients have some degree of chimerism; six of these patients, all blood transfusion-independent, have donor cells in the range of 70% to 95%, with stable mixed chimerism (MC). The seventh patient has less than 10% donor cells with, surprisingly, only minimal transfusion requirements. The detection of beta-globin gene point mutation, as used here, is a highly specific and sensitive marker for engraftment and MC in patients with thalassemia. In light of its specificity, the method is applicable in all cases of TM, as it is independent of sex and other non-globin-related DNA markers. The high incidence of MC found in our patients may be a consequence of the pre-BMT T-cell depletion. Because MC was associated with transfusion independence, complete eradication of residual host cells for effective treatment of TM and possibly other genetic diseases may prove not to be essential. PMID:7579421

  15. Expression profile of the RNA-binding protein gene hermes during chicken embryonic development.

    PubMed

    Wilmore, Helen P; McClive, Peter J; Smith, Craig A; Sinclair, Andrew H

    2005-07-01

    The hermes gene encodes an RNA-binding protein containing an RNA-recognition motif. Its expression has been described previously in Xenopus and in the developing heart of very young chicken embryos. We have analyzed the expression of cHermes in later heart development, where expression is maintained in the myocardium, and also in previously undescribed sites. cHermes expression first appears in the somites in the first terminally differentiated myocytes of both the epaxial and the hypaxial myotome. Expression is also seen in the primordium of the allantois and continues in the developing allantoic sac. cHermes expression in the pronephric and mesonephric kidneys coincides temporally and spatially with the appearance of the vascular components of the glomeruli. In addition, cHermes expression was seen in the mesoderm of the gut and in the notochord. PMID:15895363

  16. Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells.

    PubMed

    Corti, Paola; Xue, Jianmin; Tejero, Jesús; Wajih, Nadeem; Sun, Ming; Stolz, Donna B; Tsang, Michael; Kim-Shapiro, Daniel B; Gladwin, Mark T

    2016-07-26

    The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, six-coordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins. PMID:27407144

  17. PID1 (NYGGF4), a new growth-inhibitory gene in embryonal brain tumors and gliomas

    PubMed Central

    Erdreich-Epstein, Anat; Robison, Nathan; Ren, Xiuhai; Zhou, Hong; Xu, Jingying; Davidson, Tom B.; Schur, Mathew; Gilles, Floyd H.; Ji, Lingyun; Malvar, Jemily; Shackleford, Gregory M.; Margol, Ashley S.; Krieger, Mark D.; Judkins, Alexander R.; Jones, David T.W.; Pfister, Stefan; Kool, Marcel; Sposto, Richard; Asgharazadeh, Shahab

    2014-01-01

    Purpose We present here the first report of PID1 (Phosphotyrosine Interaction Domain containing 1; NYGGF4) in cancer. PID1 was identified in 2006 as a gene that modulates insulin signaling and mitochondrial function in adipocytes and muscle cells. Experimental Design and Results Using four independent medulloblastoma datasets, we show that mean PID1 mRNA levels were lower in unfavorable medulloblastomas (Groups 3 and 4, and anaplastic histology) compared with favorable medulloblastomas (SHH and WNT groups, and desmoplastic/nodular histology) and with fetal cerebellum. In two large independent glioma datasets PID1 mRNA was lower in glioblastomas (GBMs), the most malignant gliomas, compared to other astrocytomas, oligodendrogliomas and non-tumor brains. Neural and proneural GBM subtypes had higher PID1 mRNA compared to classical and mesenchymal GBM. Importantly, overall survival and radiation-free progression-free survival were longer in medulloblastoma patients with higher PID1 mRNA (univariate and multivariate analyses). Higher PID1 mRNA also correlated with longer overall survival in glioma and GBM patients. In cell culture, overexpression of PID1 inhibited colony formation in medulloblastoma, atypical teratoid rhabdoid tumor (ATRT) and GBM cell lines. Increasing PID1 also increased cell death and apoptosis, inhibited proliferation, induced mitochondrial depolarization, and decreased serum-mediated phosphorylation of AKT and ERK in medulloblastoma, ATRT and/or GBM cell lines, whereas siRNA to PID1 diminished mitochondrial depolarization. Conclusions These data are the first to link PID1 to cancer and suggest that PID1 may have a tumor inhibitory function in these pediatric and adult brain tumors. PMID:24300787

  18. Silencing of PNPLA6, the neuropathy target esterase (NTE) codifying gene, alters neurodifferentiation of human embryonal carcinoma stem cells (NT2).

    PubMed

    Pamies, D; Bal-Price, A; Fabbri, M; Gribaldo, L; Scelfo, B; Harris, G; Collotta, A; Vilanova, E; Sogorb, M A

    2014-12-01

    Neuropathy target esterase (NTE) is a protein involved in the development of a polyneuropathy caused by exposure to certain organophosphorus compounds. In vivo and in vitro studies have also associated NTE with embryonic development since NTE null mice embryos are non-viable, and silencing the NTE-codifying gene (Pnpla6) in mouse embryonic stem cells strongly alters the differentiation of vascular and nervous systems. In this paper, human embryonal carcinoma stem cells human-derived NTera2/D1 (hNT2) are used as an in vitro neurodifferentiation model to determine whether PNPLA6 silencing is able to alter the differentiation process. In control cultures, PNPLA6 mRNA levels increased in parallel with other neuroectodermal markers during neurodifferentiation. PNPLA6 silencing with specific interference RNA reached a 97% decrease in gene expression 3days after transfection and with a maximum reduction in NTE enzymatic activity (50%), observed on day 4. Silencing PNPLA6 showed an 80% decrease in quantifiable neuronal cells after 13days in vitro (DIV) compared to controls and absence of different neuronal markers after 66DIV. Microarray data analysis of the PNPLA6-silenced cells showed alterations in several developmental processes, mainly neurogenesis and epithelium tube morphogenesis. PNPLA6 silencing also led to a reduction in electrical activity and an altered neuronal phenotype. This work is the first proof supporting the hypothesis that NTE plays a role in human early neurodevelopment using a human cell differentiation model. PMID:25255935

  19. The gene for erythropoietin receptor is expressed in multipotential hematopoietic and embryonal stem cells: evidence for differentiation stage-specific regulation.

    PubMed Central

    Heberlein, C; Fischer, K D; Stoffel, M; Nowock, J; Ford, A; Tessmer, U; Stocking, C

    1992-01-01

    The principal regulator of erythropoiesis is the glycoprotein erythropoietin, which interacts with a specific cell surface receptor (EpoR). A study aimed at analyzing EpoR gene regulation has shown that both pluripotent embryonal stem cells and early multipotent hematopoietic cells express EpoR transcripts. Commitment to nonerythroid lineages (e.g., macrophage or lymphocytic) results in the shutdown of EpoR gene expression, whereas commitment to the erythroid lineage is concurrent with or followed by dramatic increases in EpoR transcription. To determine whether gene activity could be correlated with chromatin alterations, DNase-hypersensitive sites (HSS) were mapped. Two major HSS located in the promoter region and within the first intron of the EpoR gene are present in all embryonal stem and hematopoietic cells tested, the intensities of which correlate well with EpoR expression levels. In addition, a third major HSS also located within the first intron of the EpoR gene is uniquely present in erythroid cells that express high levels of EpoR. Transfection assays show that sequences surrounding this major HSS impart erythroid cell-specific enhancer activity to a heterologous promoter and that this activity is at least in part mediated by GATA-1. These data, together with concordant expression levels of GATA-1 and EpoR in both early multipotent hematopoietic and committed erythroid cells, support a regulatory role of the erythroid cell-specific transcription factor GATA-1 in EpoR transcription in these cells. However, the lack of significant levels of GATA-1 expression in embryonal stem cells implies an alternative regulatory mechanism of EpoR transcription in cells not committed to the hematopoietic lineage. Images PMID:1312671

  20. Globin chain synthesis in single erythroid bursts from cord blood: studies on gamma leads to beta and G gamma leads to A gamma switches.

    PubMed

    Comi, P; Giglioni, B; Ottolenghi, S; Gianni, A M; Polli, E; Barba, P; Covelli, A; Migliaccio, G; Condorelli, M; Peschle, C

    1980-01-01

    Erythroid bursts from cord or adult blood were grown in methylcellulose cultures (3 international units of erythropoietin per plate). On day 13, single bursts were picked up and reincubated for 16-24 hr with [3H]leucine. Radioactive globin chains [alpha,beta,G gamma, and A gamma (Ala-136)] were analyzed by either isoelectric focusing on polyacrylamide gels and fluorography or carboxymethylcellulose chromatography. In all cases, alpha to non-alpha globin radioactivity ratios were close to 1. In single cord blood bursts, the values of both gamma-to-beta and G gamma-to-A gamma ratios were spread over a large spectrum and further characterized by a continuous rather than a bimodal distribution. Morever, the G gamma-to-A gamma ratios demonstrated in single bursts appeared to be directly correlated with the respective gamma-to-beta ratios. These data suggest that both the gamma leads to beta and the G gamma leads to A gamma switches are mediated via mechanisms modulating the relative activities of the different genes in the non-alpha globin gene cluster rather than via selection of clones committed to the preferential synthesis of beta and A gamma globins. In contrast with the results obtained with cord blood, individual adult blood bursts synthesize a lower and hence relatively more uniform amount of gamma globin chains. PMID:6153796

  1. Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells.

    PubMed Central

    Gilmour, R S; Allan, M; Paul, J

    1984-01-01

    The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6). Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose. A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts. It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo. Images PMID:6330734

  2. Genetics Home Reference: methemoglobinemia, beta-globin type

    MedlinePlus

    ... blood cells. Specifically, it alters a molecule called hemoglobin within these cells. Hemoglobin within red blood cells attaches (binds) to oxygen ... in tissues throughout the body. Instead of normal hemoglobin, people with methemoglobinemia, beta-globin type have an ...

  3. Chromatin looping as a target for altering erythroid gene expression.

    PubMed

    Krivega, Ivan; Dean, Ann

    2016-03-01

    The β-hemoglobinopathies are the most common monogenic disorders in humans, with symptoms arising after birth when the fetal γ-globin genes are silenced and the adult β-globin gene is activated. There is a growing appreciation that genome organization and the folding of chromosomes are key determinants of gene transcription. Underlying this function is the activity of transcriptional enhancers that increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding or looping that is orchestrated by protein complexes that bind to both sites and stabilize their interaction. We find that enhancer activity can be redirected with concomitant changes in gene transcription. Both targeting the β-globin locus control region (LCR) to the γ-globin gene in adult erythroid cells by tethering and epigenetic unmasking of a silenced γ-globin gene lead to increased frequency of LCR/γ-globin contacts and reduced LCR/β-globin contacts. The outcome of these manipulations is robust, pancellular γ-globin transcription activation with a concomitant reduction in β-globin transcription. These examples show that chromosome looping may be considered a therapeutic target for gene activation in β-thalassemia and sickle cell disease. PMID:26918894

  4. Mixture of differentially tagged Tol2 transposons accelerates conditional disruption of a broad spectrum of genes in mouse embryonic stem cells

    PubMed Central

    Mayasari, N. Ika; Mukougawa, Keiko; Shigeoka, Toshiaki; Kawakami, Koichi; Kawaichi, Masashi; Ishida, Yasumasa

    2012-01-01

    Among the insertional mutagenesis techniques used in the current international knockout mouse project (KOMP) on the inactivation of all mouse genes in embryonic stem (ES) cells, random gene trapping has been playing a major role. Gene-targeting experiments have also been performed to individually and conditionally knockout the remaining ‘difficult-to-trap’ genes. Here, we show that transcriptionally silent genes in ES cells are severely underrepresented among the randomly trapped genes in KOMP. Our conditional poly(A)-trapping vector with a common retroviral backbone also has a strong bias to be integrated into constitutively transcribed genome loci. Most importantly, conditional gene disruption could not be successfully accomplished by using the retrovirus vector because of the frequent development of intra-vector deletions/rearrangements. We found that one of the cut and paste-type DNA transposons, Tol2, can serve as an ideal platform for gene-trap vectors that ensures identification and conditional disruption of a broad spectrum of genes in ES cells. We also solved a long-standing problem associated with multiple vector integration into the genome of a single cell by incorporating a mixture of differentially tagged Tol2 transposons. We believe our strategy indicates a straightforward approach to mass-production of conditionally disrupted alleles for genes in the target cells. PMID:22447447

  5. Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells

    SciTech Connect

    Kutsuzawa, K.; Chowdhury, E.H.; Nagaoka, M.; Maruyama, K.; Akiyama, Y.; Akaike, T. . E-mail: takaike@bio.titech.ac.jp

    2006-11-24

    Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

  6. Identification of Stage-Specific Gene Expression Signatures in Response to Retinoic Acid during the Neural Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    Akanuma, Hiromi; Qin, Xian-Yang; Nagano, Reiko; Win-Shwe, Tin-Tin; Imanishi, Satoshi; Zaha, Hiroko; Yoshinaga, Jun; Fukuda, Tomokazu; Ohsako, Seiichiroh; Sone, Hideko

    2012-01-01

    We have previously established a protocol for the neural differentiation of mouse embryonic stem cells (mESCs) as an efficient tool to evaluate the neurodevelopmental toxicity of environmental chemicals. Here, we described a multivariate bioinformatic approach to identify the stage-specific gene sets associated with neural differentiation of mESCs. We exposed mESCs (B6G-2 cells) to 10−8 or 10−7 M of retinoic acid (RA) for 4 days during embryoid body formation and then performed morphological analysis on day of differentiation (DoD) 8 and 36, or genomic microarray analysis on DoD 0, 2, 8, and 36. Three gene sets, namely a literature-based gene set (set 1), an analysis-based gene set (set 2) using self-organizing map and principal component analysis, and an enrichment gene set (set 3), were selected by the combined use of knowledge from literatures and gene information selected from the microarray data. A gene network analysis for each gene set was then performed using Bayesian statistics to identify stage-specific gene expression signatures in response to RA during mESC neural differentiation. Our results showed that RA significantly increased the size of neurosphere, neuronal cells, and glial cells on DoD 36. In addition, the gene network analysis showed that glial fibrillary acidic protein, a neural marker, remarkably up-regulates the other genes in gene set 1 and 3, and Gbx2, a neural development marker, significantly up-regulates the other genes in gene set 2 on DoD 36 in the presence of RA. These findings suggest that our protocol for identification of developmental stage-specific gene expression and interaction is a useful method for the screening of environmental chemical toxicity during neurodevelopmental periods. PMID:22891073

  7. Expression of biomarker genes of differentiation in D3 mouse embryonic stem cells after exposure to different embryotoxicant and non-embryotoxicant model chemicals

    PubMed Central

    Romero, Andrea C.; del Río, Eva; Vilanova, Eugenio; Sogorb, Miguel A.

    2015-01-01

    There is a necessity to develop in vitro methods for testing embryotoxicity (Romero et al., 2015) [1]. We studied the progress of D3 mouse embryonic stem cells differentiation exposed to model embryotoxicants and non-embryotoxicants chemicals through the expression of biomarker genes. We studied a set of 16 different genes biomarkers of general cellular processes (Cdk1, Myc, Jun, Mixl, Cer and Wnt3), ectoderm formation (Nrcam, Nes, Shh and Pnpla6), mesoderm formation (Mesp1, Vegfa, Myo1e and Hdac7) and endoderm formation (Flk1 and Afp). We offer dose response in order to derive the concentration causing either 50% or 200% of expression of the biomarker gene. These records revealed to be a valuable end-point to predict in vitro the embryotoxicity of chemicals (Romero et al., 2015) [1]. PMID:26568980

  8. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells

    SciTech Connect

    Wang Beibei; Lu Rui; Wang Weicheng; Jin Ying . E-mail: yjin@sibs.ac.cn

    2006-09-08

    The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present First application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation.

  9. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

    EPA Science Inventory

    The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

  10. Cloning and molecular characterization of an ornithine cecarboxylase gene and its expression during embryonic eevelopment of the housefly, Musca domestica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are interested in identifying targets that may be used to develop new control products for the common house fly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a cri...

  11. Single nucleotide polymorphisms in candidate genes associated with fertilizing ability of sperm and subsequent embryonic development in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fertilization and development of the preimplantation embryo is under genetic control. The goal of the current study was to test 434 single nucleotide polymorphisms (SNPs) for association with genetic variation in fertilization and early embryonic development. The approach was to produce embryos from...

  12. Involvement of histone demethylase LSD1 in short-time-scale gene expression changes during cell cycle progression in embryonic stem cells.

    PubMed

    Nair, Venugopalan D; Ge, Yongchao; Balasubramaniyan, Natarajan; Kim, Jaeyun; Okawa, Yuya; Chikina, Maria; Troyanskaya, Olga; Sealfon, Stuart C

    2012-12-01

    The histone demethylase LSD1, a component of the CoREST (corepressor for element 1-silencing transcription factor) corepressor complex, plays an important role in the downregulation of gene expression during development. However, the activities of LSD1 in mediating short-time-scale gene expression changes have not been well understood. To reveal the mechanisms underlying these two distinct functions of LSD1, we performed genome-wide mapping and cellular localization studies of LSD1 and its dimethylated histone 3 lysine 4 (substrate H3K4me2) in mouse embryonic stem cells (ES cells). Our results showed an extensive overlap between the LSD1 and H3K4me2 genomic regions and a correlation between the genomic levels of LSD1/H3K4me2 and gene expression, including many highly expressed ES cell genes. LSD1 is recruited to the chromatin of cells in the G(1)/S/G(2) phases and is displaced from the chromatin of M-phase cells, suggesting that LSD1 or H3K4me2 alternatively occupies LSD1 genomic regions during cell cycle progression. LSD1 knockdown by RNA interference or its displacement from the chromatin by antineoplastic agents caused an increase in the levels of a subset of LSD1 target genes. Taken together, these results suggest that cell cycle-dependent association and dissociation of LSD1 with chromatin mediates short-time-scale gene expression changes during embryonic stem cell cycle progression. PMID:23028048

  13. Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells

    PubMed Central

    Ge, Yongchao; Balasubramaniyan, Natarajan; Kim, Jaeyun; Okawa, Yuya; Chikina, Maria; Troyanskaya, Olga; Sealfon, Stuart C.

    2012-01-01

    The histone demethylase LSD1, a component of the CoREST (corepressor for element 1-silencing transcription factor) corepressor complex, plays an important role in the downregulation of gene expression during development. However, the activities of LSD1 in mediating short-time-scale gene expression changes have not been well understood. To reveal the mechanisms underlying these two distinct functions of LSD1, we performed genome-wide mapping and cellular localization studies of LSD1 and its dimethylated histone 3 lysine 4 (substrate H3K4me2) in mouse embryonic stem cells (ES cells). Our results showed an extensive overlap between the LSD1 and H3K4me2 genomic regions and a correlation between the genomic levels of LSD1/H3K4me2 and gene expression, including many highly expressed ES cell genes. LSD1 is recruited to the chromatin of cells in the G1/S/G2 phases and is displaced from the chromatin of M-phase cells, suggesting that LSD1 or H3K4me2 alternatively occupies LSD1 genomic regions during cell cycle progression. LSD1 knockdown by RNA interference or its displacement from the chromatin by antineoplastic agents caused an increase in the levels of a subset of LSD1 target genes. Taken together, these results suggest that cell cycle-dependent association and dissociation of LSD1 with chromatin mediates short-time-scale gene expression changes during embryonic stem cell cycle progression. PMID:23028048

  14. Comparison of gene expression regulation in mouse- and human embryonic stem cell assays during neural differentiation and in response to valproic acid exposure.

    PubMed

    Schulpen, Sjors H W; Theunissen, Peter T; Pennings, Jeroen L A; Piersma, Aldert H

    2015-08-15

    Embryonic stem cell tests (EST) are considered promising alternative assays for developmental toxicity testing. Classical mouse derived assays (mEST) are being replaced by human derived assays (hEST), in view of their relevance for human hazard assessment. We have compared mouse and human neural ESTn assays for neurodevelopmental toxicity as to regulation of gene expression during cell differentiation in both assays. Commonalities were observed in a range of neurodevelopmental genes and gene ontology (GO) terms. The mESTn showed a higher specificity in neurodevelopment than the hESTn, which may in part be caused by necessary differences in test protocols. Moreover, gene expression responses to the anticonvulsant and human teratogen valproic acid were compared. Both assays detected pharmacological and neurodevelopmental gene sets regulated by valproic acid. Common significant expression changes were observed in a subset of homologous neurodevelopmental genes. We suggest that these genes and related GO terms may provide good candidates for robust biomarkers of neurodevelopmental toxicity in hESTn. PMID:26072468

  15. Effect of egg weight on composition, embryonic growth, and expression of amino acid transporter genes in yolk sac membranes and small intestines of the domestic pigeon (Columba livia).

    PubMed

    Chen, M X; Li, X G; Yan, H C; Wang, X Q; Gao, C Q

    2016-06-01

    The objective of this study was to investigate the effect of egg weight on the composition of the egg, the growth of the embryo, and the expression of amino acid transporter genes in the yolk sac membranes and small intestines of the domestic pigeon (Columba livia). A total of 240 fertilized eggs were collected and divided into two groups based on the weight of the eggs, light (LE) and heavy (HE). The composition of 20 eggs from each group was measured, and the remaining eggs were weighed and placed in an incubator. On embryonic days (E) 9, 11, 13, and 15 and day of hatch (DOH), 15 embryos/hatchlings from each group were measured for embryonic growth, and samples were collected. The HE had heavier yolk and albumen weights than the LE (P < 0.01). Compared with the LE, the HE had heavier yolk-free embryonic body and yolk sac weights from E13 to DOH (P < 0.05). Additionally, the HE had larger yolk sac membrane weights from E13 to E15 (P < 0.05) and had more residual yolk sac content on DOH than those of the LE (P < 0.01). The yolk absorption was greater for the HE than for the LE from E11 to E13 (P < 0.05). Furthermore, the abundance of CAT2 and PepT1 mRNA in the yolk sac membranes was greater in the HE than in the LE on E13 (P < 0.05). Compared with the LE, the gene expression of EAAT2 in the intestine on E13 was greater in the HE, whereas the expression of EAAT3 was lower in the HE (P < 0.05). Taken together, our results suggest that egg weight influenced the composition of the eggs, embryonic development, and expression of amino acid transporter genes in the yolk sac membranes and small intestines of pigeon embryos. PMID:26957627

  16. Polymorphism and divergence in the beta-globin replication origin initiation region.

    PubMed

    Fullerton, S M; Bond, J; Schneider, J A; Hamilton, B; Harding, R M; Boyce, A J; Clegg, J B

    2000-01-01

    DNA sequence polymorphism and divergence was examined in the vicinity of the human beta-globin gene cluster origin of replication initiation region (IR), a 1.3-kb genomic region located immediately 5' of the adult-expressed beta-globin gene. DNA sequence variation in the replication origin IR and 5 kb of flanking DNA was surveyed in samples drawn from two populations, one African (from the Gambia, West Africa) and the other European (from Oxford, England). In these samples, levels of nucleotide and length polymorphism in the IR were found to be more than two times as high as adjacent non-IR-associated regions (estimates of per-nucleotide heterozygosity were 0.30% and 0.12%, respectively). Most polymorphic positions identified in the origin IR fall within or just adjacent to a 52-bp alternating purine-pyrimidine ((RY)n) sequence repeat. Within- and between-populations divergence is highest in this portion of the IR, and interspecific divergence in the same region, determined by comparison with an orthologous sequence from the chimpanzee, is also pronounced. Higher levels of diversity in this subregion are not, however, primarily attributable to slippage-mediated repeat unit changes, as nucleotide substitution contributes disproportionately to allelic heterogeneity. An estimate of helical stability in the sequenced region suggests that the hypervariable (RY)n constitutes the major DNA unwinding element (DUE) of the replication origin IR, the location at which the DNA duplex first unwinds and new strand synthesis begins. These findings suggest that the beta-globin IR experiences a higher underlying rate of neutral mutation than do adjacent genomic regions and that enzyme fidelity associated with the initiation of DNA replication at this origin may be compromised. The significance of these findings for our understanding of eukaryotic replication origin biology is discussed. PMID:10666717

  17. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

  18. Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia.

    PubMed

    Origa, Raffaella; Paglietti, Maria E; Sollaino, Maria C; Desogus, Maria F; Barella, Susanna; Loi, Daniela; Galanello, Renzo

    2014-01-01

    α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype-phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present. PMID:23896219

  19. Different Sets of Post-Embryonic Development Genes Are Conserved or Lost in Two Caryophyllales Species (Reaumuria soongorica and Agriophyllum squarrosum).

    PubMed

    Zhao, Pengshan; Zhang, Jiwei; Zhao, Xin; Chen, Guoxiong; Ma, Xiao-Fei

    2016-01-01

    Reaumuria soongorica and sand rice (Agriophyllum squarrosum) belong to the clade of Caryophyllales and are widely distributed in the desert regions of north China. Both plants have evolved many specific traits and adaptation strategies to cope with recurring environmental threats. However, the genetic basis that underpins their unique traits and adaptation remains unknown. In this study, the transcriptome data of R. soongorica and sand rice were compared with three other species with previously sequenced genomes (Arabidopsis thaliana, Oryza sativa, and Beta vulgaris). Four different gene sets were identified, namely, the genes conserved in both species, those lost in both species, those conserved in R. soongorica only, and those conserved in sand rice only. Gene ontology showed that post-embryonic development genes (PEDGs) were enriched in all gene sets, and different sets of PEDGs were conserved or lost in both the R. soongorica and sand rice genomes. Expression profiles of Arabidopsis orthologs further provided some clues to the function of the species-specific conserved PEDGs. Such orthologs included LEAFY PETIOLE, which could be a candidate gene involved in the development of branch priority in sand rice. PMID:26815143

  20. Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.

    PubMed Central

    Koller, B H; Kim, H S; Latour, A M; Brigman, K; Boucher, R C; Scambler, P; Wainwright, B; Smithies, O

    1991-01-01

    A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possibly passage number, influences the recovery of CFTR-targeted cells. Images PMID:1720548

  1. Different Sets of Post-Embryonic Development Genes Are Conserved or Lost in Two Caryophyllales Species (Reaumuria soongorica and Agriophyllum squarrosum)

    PubMed Central

    Zhao, Pengshan; Zhang, Jiwei; Zhao, Xin; Chen, Guoxiong; Ma, Xiao-Fei

    2016-01-01

    Reaumuria soongorica and sand rice (Agriophyllum squarrosum) belong to the clade of Caryophyllales and are widely distributed in the desert regions of north China. Both plants have evolved many specific traits and adaptation strategies to cope with recurring environmental threats. However, the genetic basis that underpins their unique traits and adaptation remains unknown. In this study, the transcriptome data of R. soongorica and sand rice were compared with three other species with previously sequenced genomes (Arabidopsis thaliana, Oryza sativa, and Beta vulgaris). Four different gene sets were identified, namely, the genes conserved in both species, those lost in both species, those conserved in R. soongorica only, and those conserved in sand rice only. Gene ontology showed that post-embryonic development genes (PEDGs) were enriched in all gene sets, and different sets of PEDGs were conserved or lost in both the R. soongorica and sand rice genomes. Expression profiles of Arabidopsis orthologs further provided some clues to the function of the species-specific conserved PEDGs. Such orthologs included LEAFY PETIOLE, which could be a candidate gene involved in the development of branch priority in sand rice. PMID:26815143

  2. Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells

    SciTech Connect

    Koller, B.H.; Hyungsuk Kim; Latour, A.M.; Brigman, K.; Boucher, R.C. Jr.; Smithies, O. ); Scambler, P.; Wainwright, B. )

    1991-12-01

    A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.

  3. Cooperativeness of the Higher Chromatin Structure of the β-Globin Locus Revealed by the Deletion Mutations of DNase I Hypersensitive site 3 of the LCR

    PubMed Central

    Fang, Xiangdong; Xiang, Ping; Yin, Wenxuan; Stamatoyannopoulos, George; Li, Qiliang

    2010-01-01

    High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5′ hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3′HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the β-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the β-globin gene and 3′HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex. PMID:17056066

  4. Identification of a Novel β-Globin Mutation (HBB: C.189_195delTCATGGC) in a Chinese Family.

    PubMed

    He, Sheng; Lin, Li; Wei, Yuan; Chen, Biyan; Yi, Shang; Chen, Qiuli; Qiu, XiaoXia; Wei, Hongwei; Li, Guojian; Zheng, Chenguang

    2016-08-01

    β-Thalassemia (β-thal) is one of the most common genetic disorders worldwide. Molecular characterization of β-thal is essential for prevention and understanding the biology of the disease. More and more rare and novel mutations are being reported. Here, we report a novel 7 bp deletion at codons 63-65 (HBB: c.189_195delTCATGGC) in exon 2 of the β-globin gene in a family from Guangxi Province, China. This novel mutation causes a shift in the normal reading frame of the β-globin coding sequence and created a stop codon at codon 87 in exon 2, which leads to a β(0)-thal phenotype. PMID:27492766

  5. Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

    PubMed Central

    Motohashi, Tsutomu; Watanabe, Natsuki; Nishioka, Masahiro; Nakatake, Yuhki; Yulan, Piao; Mochizuki, Hiromi; Kawamura, Yoshifumi; Ko, Minoru S. H.; Goshima, Naoki; Kunisada, Takahiro

    2016-01-01

    ABSTRACT Neural crest cells (NC cells) are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs) into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+) cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells. PMID:26873953

  6. Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells.

    PubMed

    Motohashi, Tsutomu; Watanabe, Natsuki; Nishioka, Masahiro; Nakatake, Yuhki; Yulan, Piao; Mochizuki, Hiromi; Kawamura, Yoshifumi; Ko, Minoru S H; Goshima, Naoki; Kunisada, Takahiro

    2016-01-01

    Neural crest cells (NC cells) are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs) into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+) cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells. PMID:26873953

  7. 6-OHDA-Induced Changes in Parkinson's Disease-Related Gene Expression are not Affected by the Overexpression of PGAM5 in In Vitro Differentiated Embryonic Mesencephalic Cells.

    PubMed

    Stępkowski, Tomasz Maciej; Wasyk, Iwona; Grzelak, Agnieszka; Kruszewski, Marcin

    2015-11-01

    LUHMES cells, a recently established line of immortalized embryonic mesencephalic cells, are the novel in vitro model for studying Parkinson's disease (PD) and dopaminergic neuron biology. Phosphoglyceromutase 5 (PGAM5) is a mitochondrial protein involved in mitophagy, mitochondria dynamics, and other processes important for PD pathogenesis. We tested the impact of lentiviral overexpression of PGAM5 protein in LUHMES cells on their differentiation and expression of 84 PD-related genes. LUHMES cells were transduced with PGAM5 or mock and treated with 100 μM 6-hydroxydopamine (6-OHDA), a model PD neurotoxin. Real-Time PCR analysis revealed that the treatment with 6-OHDA-induced changes in expression of 44 PD-related genes. PGAM5 transduction alone did not cause alternations in PD-related genes expression, nor it affected changes in gene expression mediated by 6-OHDA. The 6-OHDA-induced PD-related gene expression profile of LUHMES cells is presented for the first time and widely discussed. PMID:25986246

  8. Hypomorphic mutation of the TALE gene Prep1 (pKnox1) causes a major reduction of Pbx and Meis proteins and a pleiotropic embryonic phenotype.

    PubMed

    Ferretti, Elisabetta; Villaescusa, J Carlos; Di Rosa, Patrizia; Fernandez-Diaz, Luis C; Longobardi, Elena; Mazzieri, Roberta; Miccio, Annarita; Micali, Nicola; Selleri, Licia; Ferrari, Giuliana; Blasi, Francesco

    2006-08-01

    The interaction of Prep1 and Pbx homeodomain transcription factors regulates their activity, nuclear localization, and likely, function in development. To understand the in vivo role of Prep1, we have analyzed an embryonic lethal hypomorphic mutant mouse (Prep1(i/i)). Prep1(i/i) embryos die at embryonic day 17.5 (E17.5) to birth with an overall organ hypoplasia, severe anemia, impaired angiogenesis, and eye anomalies, particularly in the lens and retina. The anemia correlates with delayed differentiation of erythroid progenitors and may be, at least in part, responsible for intrauterine death. At E14.5, Prep1 is present in fetal liver (FL) cMyb-positive cells, whose deficiency causes a marked hematopoietic phenotype. Prep1 is also localized to FL endothelial progenitors, consistent with the observed angiogenic phenotype. Likewise, at the same gestational day, Prep1 is present in the eye cells that bear Pax6, implicated in eye development. The levels of cMyb and Pax6 in FL and in the retina, respectively, are significantly decreased in Prep1(i/i) embryos, consistent with the hematopoietic and eye phenotypes. Concomitantly, Prep1 deficiency results in the overall decrease of protein levels of its related family member Meis1 and its partners Pbx1 and Pbx2. As both Prep1 and Meis interact with Pbx, the overall Prep1/Meis-Pbx DNA-binding activity is strongly reduced in whole Prep1(i/i) embryos and their organs. Our data indicate that Prep1 is an essential gene that acts upstream of and within a Pbx-Meis network that regulates multiple aspects of embryonic development. PMID:16847320

  9. Lead induces similar gene expression changes in brains of gestationally exposed adult mice and in neurons differentiated from mouse embryonic stem cells.

    PubMed

    Sánchez-Martín, Francisco Javier; Fan, Yunxia; Lindquist, Diana M; Xia, Ying; Puga, Alvaro

    2013-01-01

    Exposure to environmental toxicants during embryonic life causes changes in the expression of developmental genes that may last for a lifetime and adversely affect the exposed individual. Developmental exposure to lead (Pb), an ubiquitous environmental contaminant, causes deficits in cognitive functions and IQ, behavioral effects, and attention deficit hyperactivity disorder (ADHD). Long-term effects observed after early life exposure to Pb include reduction of gray matter, alteration of myelin structure, and increment of criminal behavior in adults. Despite growing research interest, the molecular mechanisms responsible for the effects of lead in the central nervous system are still largely unknown. To study the molecular changes due to Pb exposure during neurodevelopment, we exposed mice to Pb in utero and examined the expression of neural markers, neurotrophins, transcription factors and glutamate-related genes in hippocampus, cortex, and thalamus at postnatal day 60. We found that hippocampus was the area where gene expression changes due to Pb exposure were more pronounced. To recapitulate gestational Pb exposure in vitro, we differentiated mouse embryonic stem cells (ESC) into neurons and treated ESC-derived neurons with Pb for the length of the differentiation process. These neurons expressed the characteristic neuronal markers Tubb3, Syp, Gap43, Hud, Ngn1, Vglut1 (a marker of glutamatergic neurons), and all the glutamate receptor subunits, but not the glial marker Gafp. Importantly, several of the changes observed in Pb-exposed mouse brains in vivo were also observed in Pb-treated ESC-derived neurons, including those affecting expression of Ngn1, Bdnf exon IV, Grin1, Grin2D, Grik5, Gria4, and Grm6. We conclude that our ESC-derived model of toxicant exposure during neural differentiation promises to be a useful model to analyze mechanisms of neurotoxicity induced by Pb and other environmental agents. PMID:24260418

  10. Lead Induces Similar Gene Expression Changes in Brains of Gestationally Exposed Adu