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Sample records for homeodomain protein yox1p

  1. The Fission Yeast Homeodomain Protein Yox1p Binds to MBF and Confines MBF-Dependent Cell-Cycle Transcription to G1-S via Negative Feedback

    PubMed Central

    Aligianni, Sofia; Lackner, Daniel H.; Klier, Steffi; Rustici, Gabriella; Wilhelm, Brian T.; Marguerat, Samuel; Codlin, Sandra; Brazma, Alvis; de Bruin, Robertus A. M.; Bähler, Jürg

    2009-01-01

    The regulation of the G1- to S-phase transition is critical for cell-cycle progression. This transition is driven by a transient transcriptional wave regulated by transcription factor complexes termed MBF/SBF in yeast and E2F-DP in mammals. Here we apply genomic, genetic, and biochemical approaches to show that the Yox1p homeodomain protein of fission yeast plays a critical role in confining MBF-dependent transcription to the G1/S transition of the cell cycle. The yox1 gene is an MBF target, and Yox1p accumulates and preferentially binds to MBF-regulated promoters, via the MBF components Res2p and Nrm1p, when they are transcriptionally repressed during the cell cycle. Deletion of yox1 results in constitutively high transcription of MBF target genes and loss of their cell cycle–regulated expression, similar to deletion of nrm1. Genome-wide location analyses of Yox1p and the MBF component Cdc10p reveal dozens of genes whose promoters are bound by both factors, including their own genes and histone genes. In addition, Cdc10p shows promiscuous binding to other sites, most notably close to replication origins. This study establishes Yox1p as a new regulatory MBF component in fission yeast, which is transcriptionally induced by MBF and in turn inhibits MBF-dependent transcription. Yox1p may function together with Nrm1p to confine MBF-dependent transcription to the G1/S transition of the cell cycle via negative feedback. Compared to the orthologous budding yeast Yox1p, which indirectly functions in a negative feedback loop for cell-cycle transcription, similarities but also notable differences in the wiring of the regulatory circuits are evident. PMID:19714215

  2. The Homeodomain Resource: a comprehensive collection of sequence, structure, interaction, genomic and functional information on the homeodomain protein family

    PubMed Central

    Moreland, R. Travis; Ryan, Joseph F.; Pan, Christopher; Baxevanis, Andreas D.

    2009-01-01

    The Homeodomain Resource is a curated collection of sequence, structure, interaction, genomic and functional information on the homeodomain family. The current version builds upon previous versions by the addition of new, complete sets of homeodomain sequences from fully sequenced genomes, the expansion of existing curated homeodomain information and the improvement of data accessibility through better search tools and more complete data integration. This release contains 1534 full-length homeodomain-containing sequences, 93 experimentally derived homeodomain structures, 101 homeodomain protein–protein interactions, 107 homeodomain DNA-binding sites and 206 homeodomain proteins implicated in human genetic disorders. Database URL: The Homeodomain Resource is freely available and can be accessed at http://research.nhgri.nih.gov/homeodomain/ PMID:20157477

  3. Pbx homeodomain proteins pattern both the zebrafish retina and tectum.

    PubMed

    French, Curtis R; Erickson, Timothy; Callander, Davon; Berry, Karyn M; Koss, Ron; Hagey, Daniel W; Stout, Jennifer; Wuennenberg-Stapleton, Katrin; Ngai, John; Moens, Cecilia B; Waskiewicz, Andrew J

    2007-07-16

    Pbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined. Analysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth. These data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth.

  4. Pbx homeodomain proteins pattern both the zebrafish retina and tectum

    PubMed Central

    French, Curtis R; Erickson, Timothy; Callander, Davon; Berry, Karyn M; Koss, Ron; Hagey, Daniel W; Stout, Jennifer; Wuennenberg-Stapleton, Katrin; Ngai, John; Moens, Cecilia B; Waskiewicz, Andrew J

    2007-01-01

    Background Pbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined. Results Analysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth. Conclusion These data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth. PMID:17634100

  5. Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

    PubMed Central

    Catron, K M; Iler, N; Abate, C

    1993-01-01

    Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes. Images PMID:8096059

  6. Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites.

    PubMed Central

    Beachy, P A; Varkey, J; Young, K E; von Kessler, D P; Sun, B I; Ekker, S C

    1993-01-01

    Cooperativity in binding of regulatory proteins to multiple DNA sites can heighten the sensitivity and specificity of the transcriptional response. We report here the cooperative DNA-binding properties of a developmentally active regulatory protein encoded by the Drosophila homeotic gene Ultrabithorax (Ubx). We show that naturally occurring binding sites for the Ubx-encoded protein contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more Ubx-encoded protein molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation in a simple yeast assay system. We found that cooperative binding is dependent on sequences outside the homeodomain, and we have identified regions predicted to form coiled coils carboxy terminal to the homeodomains of the Ubx-encoded protein and several other homeotic proteins. On the basis of our findings, we propose a multisite integrative model of homeotic protein action in which functional regulatory elements can be built from a few high-affinity sites, from many lower-affinity sites, or from sites of some intermediate number and affinity. An important corollary of this model is that even small differences in binding of homeotic proteins to individual sites could be summed to yield large overall differences in binding to multiple sites. This model is consistent with reports that homeodomain protein targets contain multiple individual binding site sequences distributed throughout sizable DNA regions. Also consistent is a recent report that sequences carboxy terminal to the Ubx homeodomain can contribute to segmental specificity. Images PMID:8105373

  7. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    PubMed

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.

  8. Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity.

    PubMed Central

    Johnson, J D; Zhang, W; Rudnick, A; Rutter, W J; German, M S

    1997-01-01

    LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. We have used yeast genetic systems to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues. PMID:9199284

  9. Homeodomain Protein Scr Regulates the Transcription of Genes Involved in Juvenile Hormone Biosynthesis in the Silkworm

    PubMed Central

    Meng, Meng; Liu, Chun; Peng, Jian; Qian, Wenliang; Qian, Heying; Tian, Ling; Li, Jiarui; Dai, Dandan; Xu, Anying; Li, Sheng; Xia, Qingyou; Cheng, Daojun

    2015-01-01

    The silkworm Dominant trimolting (Moltinism, M3) mutant undergoes three larval molts and exhibits precocious metamorphosis. In this study, we found that compared with the wild-type (WT) that undergoes four larval molts, both the juvenile hormone (JH) concentration and the expression of the JH-responsive gene Krüppel homolog 1 (Kr-h1) began to be greater in the second instar of the M3 mutant. A positional cloning analysis revealed that only the homeodomain transcription factor gene Sex combs reduced (Scr) is located in the genomic region that is tightly linked to the M3 locus. The expression level of the Scr gene in the brain-corpora cardiaca-corpora allata (Br-CC-CA) complex, which controls the synthesis of JH, was very low in the final larval instar of both the M3 and WT larvae, and exhibited a positive correlation with JH titer changes. Importantly, luciferase reporter analysis and electrophoretic mobility shift assay (EMSA) demonstrated that the Scr protein could promote the transcription of genes involved in JH biosynthesis by directly binding to the cis-regulatory elements (CREs) of homeodomain protein on their promoters. These results conclude that the homeodomain protein Scr is transcriptionally involved in the regulation of JH biosynthesis in the silkworm. PMID:26540044

  10. The Iroquois homeodomain proteins are required to specify body wall identity in Drosophila

    PubMed Central

    del Corral, Ruth Diez; Aroca, Pilar; Gómez-Skarmeta, José Luis; Cavodeassi, Florencia; Modolell, Juan

    1999-01-01

    The Iroquois complex (Iro-C) homeodomain proteins allow cells at the proximal part of the Drosophila imaginal wing disc to form mesothoracic body wall (notum). Cells lacking these proteins form wing hinge structures instead (tegula and axillary sclerites). Moreover, the mutant cells impose on neighboring wild-type cells more distal developmental fates, like lateral notum or wing hinge. These findings support a tergal phylogenetic origin for the most proximal part of the wing and provide evidence for a novel pattern organizing center at the border between the apposed notum (Iro-C-expressing) and hinge (Iro-C-nonexpressing) cells. This border is not a cell lineage restriction boundary. PMID:10398687

  11. The human cut homeodomain protein represses transcription from the c-myc promoter.

    PubMed Central

    Dufort, D; Nepveu, A

    1994-01-01

    Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene. Images PMID:8196661

  12. Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation.

    PubMed

    Maves, Lisa; Waskiewicz, Andrew Jan; Paul, Biswajit; Cao, Yi; Tyler, Ashlee; Moens, Cecilia B; Tapscott, Stephen J

    2007-09-01

    The basic helix-loop-helix (bHLH) transcription factor Myod directly regulates gene expression throughout the program of skeletal muscle differentiation. It is not known how a Myod-driven myogenic program is modulated to achieve muscle fiber-type-specific gene expression. Pbx homeodomain proteins mark promoters of a subset of Myod target genes, including myogenin (Myog); thus, Pbx proteins might modulate the program of myogenesis driven by Myod. By inhibiting Pbx function in zebrafish embryos, we show that Pbx proteins are required in order for Myod to induce the expression of a subset of muscle genes in the somites. In the absence of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By knocking down Pbx or Myod function in combination with another bHLH myogenic factor, Myf5, we show that Pbx is required for Myod to regulate fast-muscle, but not slow-muscle, development. Furthermore, we show that Sonic hedgehog requires Myod in order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. Our results reveal that Pbx proteins modulate Myod activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH proteins to establish a specific cell-type identity.

  13. Differential DNA binding properties of three human homeodomain proteins.

    PubMed Central

    Corsetti, M T; Briata, P; Sanseverino, L; Daga, A; Airoldi, I; Simeone, A; Palmisano, G; Angelini, C; Boncinelli, E; Corte, G

    1992-01-01

    The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes. Images PMID:1357628

  14. Homeodomain Interacting Protein Kinases Modulate Hypoxic Adaptation and Chemoresistance

    DTIC Science & Technology

    2015-08-01

    established cell lines. Our objectives were to (1) ectopically express or silence HIPK2 and HIPK3 in PCa cell lines (LnCAP & LnCAP-abl) , (2) define the...small RNAs for silencing . Technical difficulties prevented our group from evaluating the impact on HIPKs in PCa as ectopic expression produced a...protein of incorrect size and silencing proved ineffective. We will resolve these issue and continue to examine HIPKs in PCa in future studies. 15

  15. Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

    PubMed Central

    Jurata, L W; Kenny, D A; Gill, G N

    1996-01-01

    LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876198

  16. Pbx Homeodomain Proteins: TALEnted regulators of Limb Patterning and Outgrowth

    PubMed Central

    Capellini, Terence D.; Zappavigna, Vincenzo; Selleri, Licia

    2011-01-01

    Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression. PMID:21416555

  17. Pbx homeodomain proteins: TALEnted regulators of limb patterning and outgrowth.

    PubMed

    Capellini, Terence D; Zappavigna, Vincenzo; Selleri, Licia

    2011-05-01

    Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression. Copyright © 2011 Wiley-Liss, Inc.

  18. Functional specificity of the homeodomain protein fushi tarazu: the role of DNA-binding specificity in vivo.

    PubMed Central

    Schier, A F; Gehring, W J

    1993-01-01

    The mechanisms determining the functional specificity of Drosophila homeodomain proteins are largely unknown. Here, the role of DNA-binding specificity for the in vivo function of the homeodomain protein fushi tarazu (ftz) is analyzed. We find that specific DNA binding is an important but not sufficient determinant of the functional specificity of ftz in vivo: The ftz DNA-binding specificity mutant ftzQ50K retains partial ftz wild-type activity in gene activation and phenotypic rescue assays. Furthermore, specificity mutations in a ftz-in vivo binding site only partially reduce enhancer activity as compared to null mutations of this site. Despite bicoid-like DNA-binding specificity ftzQ50K does not activate natural or artificial bcd target genes in the realms of ftz. These results are discussed in the light of recent observations on the mechanism of action of the yeast homeodomain protein alpha 2. Images PMID:8434005

  19. Threading analysis of prospero-type homeodomains.

    PubMed

    Banerjee-Basu, S; Landsman, D; Baxevanis, A D

    1999-01-01

    The homeodomain is a common structural motif found in many transcription factors involved in cell fate determination during development. We have used threading analysis techniques to predict whether the atypical homeodomain of prospero (pros) family members could form the three-helical homeodomain structural motif, even though these proteins are not statistically similar to canonical homeodomains as assessed by BLAST searches. Amino acid sequences of these divergent homeodomain proteins were threaded through the X-ray coordinates of the Drosophila engrailed homeodomain protein [23]. The analysis confirms that the prospero class of homeodomain proteins is indeed capable of forming the homeodomain structure despite its low degree of sequence identity to the canonical homeodomain. Energy calculations indicate that the homeodomain structure is stabilized primarily by hydrophobic interactions between residues at the helical interfaces. Although the atypical prospero-type homeodomain shows very little sequence similarity when compared to other homeodomain proteins, the critical amino acids responsible for maintaining the three-dimensional structure are highly conserved. A number of other homeodomain proteins, such as PHO2p from Saccharomyces and Pax6 from human, were also included in the threading analysis and were shown to be able to form the engrailed structure, indicating that there are no rigid overall sequence requirements for the formation of the homeodomain structural motif. Based on the threading experiments and the subsequent structural alignment, a new amino acid signature that unambiguously identifies the prospero-type proteins was deduced.

  20. Modular control of glutamatergic neuronal identity in C.elegans by distinct homeodomain proteins

    PubMed Central

    Serrano-Saiz, Esther; Poole, Richard J.; Felton, Terry; Zhang, Feifan; De La Cruz, Estanisla Daniel; Hobert, Oliver

    2013-01-01

    The choice of using one of many possible neurotransmitter systems is a critical step in defining the identity of an individual neuron type. We show here that the key defining feature of glutamatergic neurons, the vesicular glutamate transporter EAT-4/VGLUT is expressed in 38 of the 118 anatomically defined neuron classes of the C.elegans nervous system. We show that eat-4/VGLUT expression is controlled in a modular manner, with distinct cis-regulatory modules driving expression in distinct glutamatergic neuron classes. We identify 13 different transcription factors, 11 of them homeodomain proteins, that act in specific combinations in 25 different glutamatergic neuron classes to initiate and maintain eat-4/VGLUT expression. We show that the adoption of a glutamatergic phenotype is linked to the adoption of other terminal identity features of a neuron, including cotransmitter phenotypes. Examination of mouse orthologs of these homeodomain proteins resulted in the identification of mouse LHX1 as a regulator of glutamatergic neurons in the brainstem. PMID:24243022

  1. The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

    PubMed

    Bellaoui, M; Pidkowich, M S; Samach, A; Kushalappa, K; Kohalmi, S E; Modrusan, Z; Crosby, W L; Haughn, G W

    2001-11-01

    Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

  2. A collective form of cell death requires homeodomain interacting protein kinase.

    PubMed

    Link, Nichole; Chen, Po; Lu, Wan-Jin; Pogue, Kristi; Chuong, Amy; Mata, Miguel; Checketts, Joshua; Abrams, John M

    2007-08-13

    We examined post-eclosion elimination of the Drosophila wing epithelium in vivo where collective "suicide waves" promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. We leveraged this phenotype in mosaic animals to discover relevant genes and establish here that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers.

  3. The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1.

    PubMed

    Zhou, Mina I; Wang, Hongmei; Ross, Jonathan J; Kuzmin, Igor; Xu, Chengen; Cohen, Herbert T

    2002-10-18

    The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.

  4. Activity regulation of a Hox protein and a role for the homeodomain in inhibiting transcriptional activation.

    PubMed

    Li, X; Murre, C; McGinnis, W

    1999-01-04

    Hox proteins are transcription factors that assign positional identities along the body axis of animal embryos. Different Hox proteins have similar DNA-binding functions in vitro and require cofactors to achieve their biological functions. Cofactors can function by enhancement of the DNA-binding specificity of Hox proteins, as has been shown for Extradenticle (Exd). We present results supporting a novel mechanism for Hox cofactor function: regulation of transcriptional activation function. First, we provide evidence that the Hox protein Deformed (Dfd) can interact with simple DNA-binding sites in Drosophila embryos in the absence of Exd, but this binding is not sufficient for transcriptional activation of reporter genes. Secondly, either Dfd or a Dfd-VP16 hybrid mediate much stronger activation in embryos on a Dfd-Exd composite site than on a simple Dfd-binding site, even though the two sites possess similar Dfd-binding affinities. This suggests that Exd is required to release the transcriptional activation function of Dfd independently of Exd enhancement of Dfd-binding affinity on the composite site. Thirdly, transfection assays confirmed that Dfd possesses an activation domain, which is suppressed in a manner dependent on the presence of the homeodomain. The regulation of Hox transcriptional activation functions may underlie the different functional specificities of proteins belonging to this developmental patterning family.

  5. Activity regulation of a Hox protein and a role for the homeodomain in inhibiting transcriptional activation.

    PubMed Central

    Li, X; Murre, C; McGinnis, W

    1999-01-01

    Hox proteins are transcription factors that assign positional identities along the body axis of animal embryos. Different Hox proteins have similar DNA-binding functions in vitro and require cofactors to achieve their biological functions. Cofactors can function by enhancement of the DNA-binding specificity of Hox proteins, as has been shown for Extradenticle (Exd). We present results supporting a novel mechanism for Hox cofactor function: regulation of transcriptional activation function. First, we provide evidence that the Hox protein Deformed (Dfd) can interact with simple DNA-binding sites in Drosophila embryos in the absence of Exd, but this binding is not sufficient for transcriptional activation of reporter genes. Secondly, either Dfd or a Dfd-VP16 hybrid mediate much stronger activation in embryos on a Dfd-Exd composite site than on a simple Dfd-binding site, even though the two sites possess similar Dfd-binding affinities. This suggests that Exd is required to release the transcriptional activation function of Dfd independently of Exd enhancement of Dfd-binding affinity on the composite site. Thirdly, transfection assays confirmed that Dfd possesses an activation domain, which is suppressed in a manner dependent on the presence of the homeodomain. The regulation of Hox transcriptional activation functions may underlie the different functional specificities of proteins belonging to this developmental patterning family. PMID:9878063

  6. Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

    SciTech Connect

    Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Sang Ki; Lee, Sang Do; Park, Jin Bong; Chang, Seok Jong; Jeon, Byeong Hwa

    2011-07-01

    Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.

  7. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans

    PubMed Central

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-01

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development. PMID:26791749

  8. The Homeodomain Iroquois Proteins Control Cell Cycle Progression and Regulate the Size of Developmental Fields.

    PubMed

    Barrios, Natalia; González-Pérez, Esther; Hernández, Rosario; Campuzano, Sonsoles

    2015-08-01

    During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins play a key, evolutionarily conserved, role in territorial specification. Here we show that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. We demonstrate that physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, our results provide a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors.

  9. HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.

    PubMed

    Kömüves, László G; Michael, Elias; Arbeit, Jeffrey M; Ma, Xiao-Kui; Kwong, Angela; Stelnicki, Eric; Rozenfeld, Sophia; Morimune, M; Yu, Qian-Chun; Largman, Corey

    2002-05-01

    The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha-2-integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA-positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human

  10. Class III homeodomain leucine-zipper proteins regulate xylem cell differentiation.

    PubMed

    Ohashi-Ito, Kyoko; Kubo, Minoru; Demura, Taku; Fukuda, Hiroo

    2005-10-01

    Although it has been suggested that class III homeodomain leucine-zipper proteins (HD-Zip III) are involved in vascular development, details of the function of individual HD-Zip III proteins in vascular differentiation have not been resolved. To understand the function of each HD-Zip III protein in vascular differentiation precisely, we analyzed the in vitro transcriptional activity and in vivo function of Zinnia HD-Zip III genes, ZeHB-10, ZeHB-11 and ZeHB-12, which show xylem-related expression. Transgenic Arabidopsis plants harboring cauliflower mosaic virus 35S-driven ZeHB-10 and ZeHB-12 with a mutation in the START domain (mtZeHB-10, mtZeHB-12) showed a higher production of tracheary elements (TEs) and xylem precursor cells, respectively. A systematic analysis with Genechip arrays revealed that overexpression of mtZeHB-12 rapidly induced various genes, including brassinosteroid-signaling pathway-related genes and genes for transcription factors that are expressed specifically in vascular tissues in situ. Furthermore, mtZeHB-12 overexpression did not induce TE-specific genes, including genes related to programmed cell death and lignin polymerization, but did induce lignin monomer synthesis-related genes, which are expressed in xylem parenchyma cells. These results suggest that ZeHB-12 is involved in the differentiation of xylem parenchyma cells, but not of TEs.

  11. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    SciTech Connect

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45{sup +} hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45{sup low}c-Kit{sup +} cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

  12. Paired and LIM class homeodomain proteins coordinate differentiation of the C. elegans ALA neuron.

    PubMed

    Van Buskirk, Cheryl; Sternberg, Paul W

    2010-06-01

    The ancient origin of sleep is evidenced by deeply conserved signaling pathways regulating sleep-like behavior, such as signaling through the Epidermal growth factor receptor (EGFR). In Caenorhabditis elegans, a sleep-like state can be induced at any time during development or adulthood through conditional expression of LIN-3/EGF. The behavioral response to EGF is mediated by EGFR activity within a single cell, the ALA neuron, and mutations that impair ALA differentiation are expected to confer EGF-resistance. Here we describe three such EGF-resistant mutants. One of these corresponds to the LIM class homeodomain (HD) protein CEH-14/Lhx3, and the other two correspond to Paired-like HD proteins CEH-10/Chx10 and CEH-17/Phox2. Whereas CEH-14 is required for ALA-specific gene expression throughout development, the Prd-like proteins display complementary temporal contributions to gene expression, with the requirement for CEH-10 decreasing as that of CEH-17 increases. We present evidence that CEH-17 participates in a positive autoregulatory loop with CEH-14 in ALA, and that CEH-10, in addition to its role in ALA differentiation, functions in the generation of the ALA neuron. Similarly to CEH-17, CEH-10 is required for the posterior migration of the ALA axons, but CEH-14 appears to regulate an aspect of ALA axon outgrowth that is distinct from that of the Prd-like proteins. Our findings reveal partial modularity among the features of a neuronal differentiation program and their coordination by Prd and LIM class HD proteins.

  13. Functional differences between HOX proteins conferred by two residues in the homeodomain N-terminal arm.

    PubMed Central

    Phelan, M L; Sadoul, R; Featherstone, M S

    1994-01-01

    Hox genes encode homeodomain-containing transcriptional regulators that function during development to specify positional identity along embryonic axes. The homeodomain is composed of a flexible N-terminal arm and three alpha helices, and it differentially binds DNA. A number of homeodomains recognize sites containing a TAAT core motif. The product of the murine Hoxd-4 (Hox-4.2) gene functions in a positive autoregulatory fashion in P19 cells that is dependent on two TAAT motifs in the Hoxd-4 promoter. This effect is specific in that murine HOXA-1 (HOX-1.6) is unable to activate transcription through the Hoxd-4 autoregulatory element. Here we show that this is due to an inability of the HOXA-1 homeodomain to bind a HOXD-4 recognition site effectively. We have produced chimeras between HOXD-4 and HOXA-1 to map specific residues responsible for this functional difference. When positions 2 and 3 in the N-terminal arm of HOXA-1 were converted to HOXD-4 identity, both strong DNA binding and transcriptional activation were rescued. This substitution appears to confer an increased DNA-binding ability on the HOXA-1 homeodomain, since we were unable to detect a high-affinity recognition sequence for HOXA-1 in a randomized pool of DNA probes. The contribution of position 3 to DNA binding has been implicated by structural studies, but this is the first report of the importance of position 2 in regulating homeodomain-DNA interactions. Additionally, specific homeodomain residues that confer major differences in DNA binding and transcriptional activation between Hox gene products have not been previously determined. Identity at these two positions is generally conserved among paralogs but varies between Hox gene subfamilies. As a result, these residues may be important for the regulation of target gene expression by specific Hox products. Images PMID:7913516

  14. Homeodomain-interacting protein kinase 2 plays an important role in normal terminal erythroid differentiation.

    PubMed

    Hattangadi, Shilpa M; Burke, Karly A; Lodish, Harvey F

    2010-06-10

    Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as alpha-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

  15. Protein and DNA contact surfaces that mediate the selective action of the Phox1 homeodomain at the c-fos serum response element.

    PubMed Central

    Simon, K J; Grueneberg, D A; Gilman, M

    1997-01-01

    The human homeodomain protein Phox1 can impart serum-responsive transcriptional activity to the c-fos serum response element (SRE) by interacting with serum response factor (SRF). This activity is shared with other Paired class homeodomains but not with more distantly related homeodomains. To understand the mechanism of action of Phox1 at the SRE and the basis for the selective activity of Paired class homeodomains in this context, we performed a detailed mutagenesis of the Phox1 homeodomain. We found that amino acid residues that contact the major groove of the DNA are required for SRE activation in vivo, suggesting an in vivo requirement for major-groove DNA contact by the homeodomain. In contrast, substitution of a lysine residue in the N-terminal arm of the Phox1 homeodomain appeared to abolish DNA binding without affecting activity in vivo. Certain substitutions on the exposed surfaces of helices 1 and 2, not required for DNA binding, abolished activity in vivo, suggesting that these surfaces contact an accessory protein(s) required for this activity. We also found that transfer of a single amino acid residue from the surface of Phox1 helix 1 to the corresponding position in the distantly related Deformed (Dfd) homeodomain imparts to Dfd the ability to activate the SRE in vivo. We propose that Phox1 interacts with one or more factors at the SRE, in addition to SRF, and that the specificity of this interaction is determined by residues on the surfaces of helices 1 and 2. PMID:9343429

  16. Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity

    PubMed Central

    Busser, Brian W.; Shokri, Leila; Jaeger, Savina A.; Gisselbrecht, Stephen S.; Singhania, Aditi; Berger, Michael F.; Zhou, Bo; Bulyk, Martha L.; Michelson, Alan M.

    2012-01-01

    A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein-binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs that have mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To understand better the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou-binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity. PMID:22296846

  17. Members of the meis1 and pbx homeodomain protein families cooperatively bind a cAMP-responsive sequence (CRS1) from bovine CYP17.

    PubMed

    Bischof, L J; Kagawa, N; Moskow, J J; Takahashi, Y; Iwamatsu, A; Buchberg, A M; Waterman, M R

    1998-04-03

    The mammalian Pbx homeodomain proteins provide specificity and increased DNA binding affinity to other homeodomain proteins. A cAMP-responsive sequence (CRS1) from bovine CYP17 has previously been shown to be a binding site for Pbx1. A member of a second mammalian homeodomain family, Meis1, is now also demonstrated to be a CRS1-binding protein upon purification using CRS1 affinity chromatography. CRS1 binding complexes from Y1 adrenal cell nuclear extract contain both Pbx1 and Meis1. This is the first transcriptional regulatory element reported as a binding site for members of the Meis1 homeodomain family. Pbx1 and Meis1 bind cooperatively to CRS1, whereas neither protein can bind this element alone. Mutagenesis of the CRS1 element indicates a binding site for Meis1 adjacent to the Pbx site. All previously identified Pbx binding partners have Pbx interacting motifs that contain a tryptophan residue amino-terminal to the homeodomain that is required for cooperative binding to DNA with Pbx. Members of the Meis1 family contain one tryptophan residue amino-terminal to the homeodomain, but site-directed mutagenesis indicates that this residue is not required for cooperative CRS1 binding with Pbx. Thus, the Pbx-Meis1 interaction is unique among Pbx complexes. Meis1 also cooperatively binds CRS1 with the Pbx homologs extradenticle from Drosophila melanogaster and ceh-20 from Caenorhabditis elegans, indicating that this interaction is evolutionarily conserved. Thus, CYP17 CRS1 is a transcriptional regulatory element containing both Pbx and Meis1 binding sites, which permit these two homeodomain proteins to bind and potentially regulate cAMP-dependent transcription through this sequence.

  18. Specific DNA binding of the two chicken Deformed family homeodomain proteins, Chox-1.4 and Chox-a.

    PubMed Central

    Sasaki, H; Yokoyama, E; Kuroiwa, A

    1990-01-01

    The cDNA clones encoding two chicken Deformed (Dfd) family homeobox containing genes Chox-1.4 and Chox-a were isolated. Comparison of their amino acid sequences with another chicken Dfd family homeodomain protein and with those of mouse homologues revealed that strong homologies are located in the amino terminal regions and around the homeodomains. Although homologies in other regions were relatively low, some short conserved sequences were also identified. E. coli-made full length proteins were purified and used for the production of specific antibodies and for DNA binding studies. The binding profiles of these proteins to the 5'-leader and 5'-upstream sequences of Chox-1.4 and Chox-a coding regions were analyzed by immunoprecipitation and DNase I footprint assays. These two Chox proteins bound to the same sites in the 5'-flanking sequences of their coding regions with various affinities and their binding affinities to each site were nearly the same. The consensus sequences of the high and low affinity binding sites were TAATGA(C/G) and CTAATTTT, respectively. A clustered binding site was identified in the 5'-upstream of the Chox-a gene, suggesting that this clustered binding site works as a cis-regulatory element for auto- and/or cross-regulation of Chox-a gene expression. Images PMID:1970866

  19. The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

    PubMed Central

    Wolfram, Verena; Southall, Tony D.; Brand, Andrea H.; Baines, Richard A.

    2012-01-01

    Summary Neuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K+ currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K+ current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes. PMID:22920257

  20. Sequence-specific binding to telomeric DNA by CEH-37, a homeodomain protein in the nematode Caenorhabditis elegans.

    PubMed

    Kim, Seung Hyun; Hwang, Soon Baek; Chung, In Kwon; Lee, Junho

    2003-07-25

    Caenorhabditis elegans can serve as a model system to study telomere functions due to its similarity to higher organisms in telomere structures. We report here the identification of the nematode homeodomain protein CEH-37 as a telomere-binding protein using a yeast one-hybrid screen. The predicted three-dimensional model of the homeodomain of CEH-37, which has a typical helix-loop-helix structure, was similar to that of the Myb domain of known telomere-binding proteins, which is also a helix-loop-helix protein, despite little amino acid sequence similarity. We demonstrated the specific binding of CEH-37 to the nematode telomere sequences in vitro by competition assays. We determined that CEH-37 binding required at least 1.5 repeats of TTAGGC and that the core sequence for binding was GGCTTA. We found that CEH-37 had an ability to bend telomere sequence-containing DNA, which is the case for other known telomere-binding proteins such as TRF1 and RAP1, indicating that CEH-37 may be involved in establishing or maintaining a secondary structure of the telomeres in vivo. We also demonstrated that CEH-37 was primarily co-localized to the chromosome ends in vivo, indicating that CEH-37 may play roles in telomere functions. Consistent with this, a ceh-37 mutation resulting in a truncated protein caused a weak high incidence of male phenotype, which may have been caused by chromosome instability. The identification of CEH-37 as a telomere-binding protein may represent an evolutionary conservation of telomere-binding proteins in terms of tertiary protein structure rather than primary amino acid sequence.

  1. Protein Inhibitor of Activated STAT Y (PIASy) Regulates Insulin Secretion by Interacting with LIM Homeodomain Transcription Factor Isl1

    PubMed Central

    Yan, Chengzhi; Yu, Chulin; Zhang, Di; Cui, Yan; Zhou, Jinlian; Cui, Sheng

    2016-01-01

    It is known that the LIM homeodomain transcription factor Isl1 is highly expressed in all pancreatic endocrine cells and functions in regulating pancreatic development and insulin secretion. The Isl1 mutation has been found to be associated with type 2 diabetes, but the mechanism responsible for Isl1 regulation of insulin synthesis and secretion still needs to be elucidated. In the present study, the protein inhibitor of activated STAT Y (PIASy) was identified as a novel Isl1-interacting protein with a yeast two-hybrid system, and its interaction with Isl1 was further confirmed by a co-immunoprecipitation experiment. PIASy and Isl1 colocalize in human and mouse pancreas and NIT beta cells. Furthermore, PIASy and Isl1 upregulate insulin gene expression and insulin secretion in a dose-dependent manner by activating the insulin promoter. PIASy and Isl1 mRNA expression levels were also increased in type 2 diabetic db/db mice. In addition, our results demonstrate that PIASy and Isl1 cooperate to activate the insulin promoter through the Isl1 homeodomain and PIASy ring domain. These data suggest that that PIASy regulates insulin synthesis and secretion by interacting with Isl1 and provide new insight into insulin regulation, although the detailed molecular mechanism needs to be clarified in future studies. PMID:28000708

  2. Conserved homeodomain proteins interact with MADS box protein Mcm1 to restrict ECB-dependent transcription to the M/G1 phase of the cell cycle

    PubMed Central

    Pramila, Tata; Miles, Shawna; GuhaThakurta, Debraj; Jemiolo, Dave; Breeden, Linda L.

    2002-01-01

    Two homeodomain proteins, Yox1 and Yhp1, act as repressors at early cell cycle boxes (ECBs) to restrict their activity to the M/G1 phase of the cell cycle in budding yeast. These proteins bind to Mcm1 and to a typical homeodomain binding site. The expression of Yox1 is periodic and directly correlated with its binding to, and repression of, ECB activity. The absence of Yox1 and Yhp1 or the constitutive expression of Yox1 leads to the loss of cell-cycle regulation of ECB activity. Therefore, the cell-cycle-regulated expression of these repressors defines the interval of ECB-dependent transcription. Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway. PMID:12464633

  3. Conserved homeodomain proteins interact with MADS box protein Mcm1 to restrict ECB-dependent transcription to the M/G1 phase of the cell cycle.

    PubMed

    Pramila, Tata; Miles, Shawna; GuhaThakurta, Debraj; Jemiolo, Dave; Breeden, Linda L

    2002-12-01

    Two homeodomain proteins, Yox1 and Yhp1, act as repressors at early cell cycle boxes (ECBs) to restrict their activity to the M/G1 phase of the cell cycle in budding yeast. These proteins bind to Mcm1 and to a typical homeodomain binding site. The expression of Yox1 is periodic and directly correlated with its binding to, and repression of, ECB activity. The absence of Yox1 and Yhp1 or the constitutive expression of Yox1 leads to the loss of cell-cycle regulation of ECB activity. Therefore, the cell-cycle-regulated expression of these repressors defines the interval of ECB-dependent transcription. Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway.

  4. The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors

    PubMed Central

    Kikuchi, Yutaka; Trinh, Le A.; Reiter, Jeremy F.; Alexander, Jonathan; Yelon, Deborah; Stainier, Didier Y.R.

    2000-01-01

    Vertebrate endoderm development has recently become the focus of intense investigation. In this report, we first show that the zebrafish bonnie and clyde (bon) gene plays a critical early role in endoderm formation. bon mutants exhibit a profound reduction in the number of sox17-expressing endodermal precursors formed during gastrulation, and, consequently, a profound reduction in gut tissue at later stages. The endodermal precursors that do form in bon mutants, however, appear to differentiate normally indicating that bon is not required at later steps of endoderm development. We further demonstrate that bon encodes a paired-class homeodomain protein of the Mix family that is expressed transiently before and during early gastrulation in both mesodermal and endodermal progenitors. Overexpression of bon can rescue endodermal gene expression and the formation of a gut tube in bon mutants. Analysis of a newly identified mutant allele reveals that a single amino acid substitution in the DNA recognition helix of the homeodomain creates a dominant interfering form of Bon when overexpressed. We also show through loss- and gain-of-function analyses that Bon functions exclusively downstream of cyclops and squint signaling. Together, our data demonstrate that Bon is a critical transcriptional regulator of early endoderm formation. PMID:10817762

  5. Glucocorticoids repress transcription from a negative glucocorticoid response element recognized by two homeodomain-containing proteins, Pbx and Oct-1.

    PubMed

    Subramaniam, N; Cairns, W; Okret, S

    1998-09-04

    Several studies have established that the prolactin (PRL) gene is expressed not only in lactotrophs and somatotrophs of the anterior pituitary but, albeit to a lesser extent, in non-pituitary cells like human thymocytes, decidualized endometrium, mammary glands during lactation, and some human non-pituitary cell lines. Despite the requirement in the pituitary for the pituitary-specific transcription factor Pit-1/GHF-1 for PRL expression, the expression in non-pituitary cells occurs in the absence of Pit-1/GHF-1 and can be repressed by glucocorticoids. This prompted us to investigate the transcription factors in non-pituitary cells which are involved in controlling expression and glucocorticoid repression of a previously characterized negative glucocorticoid response element from the bovine prolactin gene (PRL3 nGRE). Here we have demonstrated that non-pituitary cells (COS-7 and mouse hepatoma Hepa1c1c7 cells) conferred increased expression via the PRL3 nGRE mainly because of the binding of the ubiquitously expressed POU-homeodomain-containing octamer transcription factor-1 (Oct-1) to an AT-rich sequence present in the PRL3 sequence. However, full transcriptional activity required the binding of a second ubiquitously expressed homeodomain-containing protein, Pbx, previously shown to bind cooperatively with several homeotic selector proteins. The Pbx binding site in the PRL3 nGRE, located just upstream of the Oct-1 binding site, showed a strong sequence similarity with known Pbx binding sites and bound Pbx with an affinity similar to that of other established Pbx target sequences. Interestingly, both Oct-1 and Pbx binding to the PRL3 nGRE were found to be required for glucocorticoid repression. Addition of in vitro translated glucocorticoid receptor DNA binding domain to the nuclear extract prevented Oct-1 and Pbx from binding to the PRL element. The involvement of the homeobox protein Pbx in glucocorticoid repression via an nGRE identifies a new role for this

  6. Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity

    PubMed Central

    Merelo, Paz; Ram, Hathi; Pia Caggiano, Monica; Ohno, Carolyn; Ott, Felix; Straub, Daniel; Graeff, Moritz; Cho, Seok Keun; Yang, Seong Wook; Wenkel, Stephan; Heisler, Marcus G.

    2016-01-01

    A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial–abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets. PMID:27698117

  7. Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity.

    PubMed

    Merelo, Paz; Ram, Hathi; Pia Caggiano, Monica; Ohno, Carolyn; Ott, Felix; Straub, Daniel; Graeff, Moritz; Cho, Seok Keun; Yang, Seong Wook; Wenkel, Stephan; Heisler, Marcus G

    2016-10-18

    A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial-abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets.

  8. The Proline Rich Homeodomain Protein PRH/Hhex Forms Stable Oligomers That Are Highly Resistant to Denaturation

    PubMed Central

    Shukla, Anshuman; Burton, Nicholas M.; Jayaraman, Padma-Sheela; Gaston, Kevin

    2012-01-01

    Background Many transcription factors control gene expression by binding to specific DNA sequences at or near the genes that they regulate. However, some transcription factors play more global roles in the control of gene expression by altering the architecture of sections of chromatin or even the whole genome. The ability to form oligomeric protein assemblies allows many of these proteins to manipulate extensive segments of DNA or chromatin via the formation of structures such as DNA loops or protein-DNA fibres. Principal Findings Here we show that the proline rich homeodomain protein PRH/Hhex forms predominantly octameric and/or hexadecameric species in solution as well as larger assemblies. We show that these assemblies are highly stable resisting denaturation by temperature and chemical denaturants. Conclusion These data indicate that PRH is functionally and structurally related to the Lrp/AsnC family of proteins, a group of proteins that are known to act globally to control gene expression in bacteria and archaea. PMID:22540015

  9. Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

    PubMed

    Akaike, Y; Kuwano, Y; Nishida, K; Kurokawa, K; Kajita, K; Kano, S; Masuda, K; Rokutan, K

    2015-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent

  10. Arabidopsis homeodomain-leucine zipper IV proteins promote stomatal development and ectopically induce stomata beyond the epidermis.

    PubMed

    Peterson, Kylee M; Shyu, Christine; Burr, Christian A; Horst, Robin J; Kanaoka, Masahiro M; Omae, Minami; Sato, Yutaka; Torii, Keiko U

    2013-05-01

    The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of 'master regulatory' transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

  11. Homeodomain Protein Transforming Growth Factor Beta-Induced Factor 2 Like, X-Linked Function in Colon Adenocarcinoma Cells

    PubMed

    Akbari, Abolfazl; Agah, Shahram; Heidari, Mansour; Mobini, Gholam Reza; Faghihloo, Ebrahim; Sarveazad, Arash; Mirzaei, Alireza

    2017-08-27

    Background: TGIF2LX (transforming growth factor beta-induced factor 2 like, X-linked) is a homeodomain (HD) protein that has been implicated in the negative regulation of cell signaling pathways. The aim of this study was to investigate the possible functions of TGIF2LX in colon adenocarcinoma cells. Methods: The human SW48 cell line was transfected with cDNA for the wild-type TGIF2LX gene and gene/protein over-expression was confirmed by microscopic analysis, real time RT-PCR and Western blotting techniques. In vitro cell proliferation was evaluated by MTT and BrdU assays. After developing a colon tumor model in nude mice, immunohistochemical (IHC) staining of tumor tissue was carried out for Ki-67 (proliferation) and CD34 (angiogenesis) markers. To predict potential protein partners of TGIF2LX, in-silico analysis was also conducted. Results: Obtained results showed over-expression of TGIF2LX as a potential transcription factor could inhibit either proliferation or angiogenesis (P<0.05) in colon tumors. In-silico results predicted interaction of TGIF2LX with other proteins considered important for cellular development. Conclusions: Our findings provided evidence of molecular mechanisms by which TGIF2LX could act as a tumor suppressor in colon adenocarcinoma cells. Thus, this gene may potentially be a promising option for colon cancer gene-based therapeutic strategies. Creative Commons Attribution License

  12. Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

    PubMed

    Jun, S; Wallen, R V; Goriely, A; Kalionis, B; Desplan, C

    1998-11-10

    Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

  13. Recruitment of the priming protein pTP and DNA binding occur by overlapping Oct-1 POU homeodomain surfaces

    PubMed Central

    de Jong, R.N.; Mysiak, M.E.; Meijer, L.A.T.; van der Linden, M.; van der Vliet, P.C.

    2002-01-01

    The human transcription factor Oct-1 can stimulate transcription from a variety of promoters by interacting with the coactivators OBF-1/OCA-B/BOB-1, SNAP190 and VP16. These proteins contact Oct-1 regions different from the DNA binding surface. Oct-1 also stimulates the DNA replication of adenovirus through its DNA binding site in the origin. The Oct-1 POU homeodomain (POUhd) binds the adenovirus precursor terminal protein pTP, which serves as the protein primer of DNA replication and recruits pTP to the origin. To map the interaction with pTP at the POUhd surface, we screened a library of randomly mutated POU domains and identified mutations that interfered with pTP interaction and DNA replication stimulation. These mutants clustered at a surface different from those recognized by OBF-1, SNAP190 and VP16. Unexpectedly, the pTP binding region largely overlapped with the DNA binding surface of POUhd. In agreement with this, pTP binding and DNA binding were mutually exclusive. We propose a model to reconcile pTP recruitment and DNA binding by Oct-1. PMID:11847120

  14. Phosphorylation status of the SCR homeodomain determines its functional activity: essential role for protein phosphatase 2A,B′

    PubMed Central

    Berry, Meera; Gehring, Walter

    2000-01-01

    Sex combs reduced (SCR) is a Drosophila Hox protein that determines the identity of the labial and prothoracic segments. In search of factors that might associate with SCR to control its activity and/or specificity, we performed a yeast two-hybrid screen. A Drosophila homologue of the regulatory subunit (B′/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B′) specifically interacted with the SCR homeodomain. The N-terminal arm within the SCR homeodomain was shown to be a target of phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo analyses revealed that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the homeodomain were active or inactive, respectively. Inactivity of the phosphorylated mimic form was attributed to impaired DNA binding. Specific ablation of dPP2A,B′ gene activity by double-stranded RNA-mediated genetic interference resulted in embryos without salivary glands, an SCR null phenotype. Our data demonstrate an essential role for Drosophila PP2A,B′ in positively modulating SCR function. PMID:10856239

  15. The Alfin-like homeodomain finger protein AL5 suppresses multiple negative factors to confer abiotic stress tolerance in Arabidopsis.

    PubMed

    Wei, Wei; Zhang, Yu-Qin; Tao, Jian-Jun; Chen, Hao-Wei; Li, Qing-Tian; Zhang, Wan-Ke; Ma, Biao; Lin, Qing; Zhang, Jin-Song; Chen, Shou-Yi

    2015-03-01

    Plant homeodomain (PHD) finger proteins affect processes of growth and development by changing transcription and reading epigenetic histone modifications, but their functions in abiotic stress responses remain largely unclear. Here we characterized seven Arabidopsis thaliana Alfin1-like PHD finger proteins (ALs) in terms of the responses to abiotic stresses. ALs localized to the nucleus and repressed transcription. Except AL6, all the ALs bound to G-rich elements. Mutations of the amino acids at positions 34 and 35 in AL6 caused loss of ability to bind to G-rich elements. Expression of the AL genes responded differentially to osmotic stress, salt, cold and abscisic acid treatments. AL5-over-expressing plants showed higher tolerance to salt, drought and freezing stress than Col-0. Consistently, al5 mutants showed reduced stress tolerance. We used ChIP-Seq assays to identify eight direct targets of AL5, and found that AL5 binds to the promoter regions of these genes. Knockout mutants of five of these target genes exhibited varying tolerances to stresses. These results indicate that AL5 inhibits multiple signaling pathways to confer stress tolerance. Our study sheds light on mechanisms of AL5-mediated signaling in abiotic stress responses, and provides tools for improvement of stress tolerance in crop plants.

  16. The Homeodomain Protein Defective Proventriculus Is Essential for Male Accessory Gland Development to Enhance Fecundity in Drosophila

    PubMed Central

    Minami, Ryunosuke; Wakabayashi, Miyuki; Sugimori, Seiko; Taniguchi, Kiichiro; Kokuryo, Akihiko; Imano, Takao; Adachi-Yamada, Takashi; Watanabe, Naoko; Nakagoshi, Hideki

    2012-01-01

    The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. We provide the first evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity. PMID:22427829

  17. The homeodomain protein defective proventriculus is essential for male accessory gland development to enhance fecundity in Drosophila.

    PubMed

    Minami, Ryunosuke; Wakabayashi, Miyuki; Sugimori, Seiko; Taniguchi, Kiichiro; Kokuryo, Akihiko; Imano, Takao; Adachi-Yamada, Takashi; Watanabe, Naoko; Nakagoshi, Hideki

    2012-01-01

    The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. We provide the first evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity.

  18. Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5'-flanking region.

    PubMed Central

    Yamaguchi, M; Nishida, Y; Moriuchi, T; Hirose, F; Hui, C C; Suzuki, Y; Matsukage, A

    1990-01-01

    The genomic and cDNA clones for a Drosophila melanogaster proliferating cell nuclear antigen (PCNA) (cyclin) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide was interrupted by a single short intron of 60 base pairs (bp), and about 70% of the deduced amino acid sequence of the Drosophila PCNA was identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome. The PCNA mRNA was detected at a high level in adult ovaries, unfertilized eggs, and early embryos and at low levels in the other developmental stages. The major transcription initiation site (cap site) was localized at 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box was found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence similar to the binding sites for Drosophila proteins containing homeodomains were found in the region from -127 to -413. DNase I footprint analysis revealed that the Drosophila homeodomain proteins coded by even-skipped and zerknüllt genes can specifically bind to these sites. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins. Images PMID:1968224

  19. The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures.

    PubMed

    Fekany, K; Yamanaka, Y; Leung, T; Sirotkin, H I; Topczewski, J; Gates, M A; Hibi, M; Renucci, A; Stemple, D; Radbill, A; Schier, A F; Driever, W; Hirano, T; Talbot, W S; Solnica-Krezel, L

    1999-04-01

    The dorsal gastrula organizer plays a fundamental role in establishment of the vertebrate axis. We demonstrate that the zebrafish bozozok (boz) locus is required at the blastula stages for formation of the embryonic shield, the equivalent of the gastrula organizer and expression of multiple organizer-specific genes. Furthermore, boz is essential for specification of dorsoanterior embryonic structures, including notochord, prechordal mesendoderm, floor plate and forebrain. We report that boz mutations disrupt the homeobox gene dharma. Overexpression of boz in the extraembryonic yolk syncytial layer of boz mutant embryos is sufficient for normal development of the overlying blastoderm, revealing an involvement of extraembryonic structures in anterior patterning in fish similarly to murine embryos. Epistatic analyses indicate that boz acts downstream of beta-catenin and upstream to TGF-beta signaling or in a parallel pathway. These studies provide genetic evidence for an essential function of a homeodomain protein in beta-catenin-mediated induction of the dorsal gastrula organizer and place boz at the top of a hierarchy of zygotic genes specifying the dorsal midline of a vertebrate embryo.

  20. The homeodomain protein Cux1 interacts with Grg4 to repress p27 kip1 expression during kidney development.

    PubMed

    Sharma, Madhulika; Brantley, Jennifer G; Vassmer, Dianne; Chaturvedi, Gaurav; Baas, Jennifer; Vanden Heuvel, Gregory B

    2009-06-15

    The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27(kip1) promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27(kip1) in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27(kip1) at two separate sites in the p27(kip1) promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27(kip1) promoter on the sequence containing two Sp1 sites and a CCAAT box approximately 500 bp from the transcriptional start site, and to an AT rich sequence approximately 1.5 kb from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27(kip1) repression by Cux1 during kidney development.

  1. Downregulation of homeodomain-interacting protein kinase-2 contributes to bladder cancer metastasis by regulating Wnt signaling.

    PubMed

    Tan, Mingyue; Gong, Hua; Zeng, Yigang; Tao, Le; Wang, Jun; Jiang, Juntao; Xu, Dongliang; Bao, Erdun; Qiu, Jianxin; Liu, Zhihong

    2014-10-01

    Homeodomain-interacting protein kinase-2 (Hipk2) has been shown to have important regulatory roles in cancer biology, such as cancer cell proliferation, cell cycle, and cell invasion. However, the contributions of Hipk2 to bladder cancer metastasis remain largely unknown. In the current study, we assayed the expression level of Hipk2 in bladder cancer tissues by real-time PCR, and defined its biological functions. We found that Hipk2 levels were downregulated in most bladder cancer tissues compared with adjacent normal tissues, and Hipk2 levels were remarkably decreased in metastasized tumor tissues when compared with primary tumors. SiRNA-mediated Hipk2 silencing increased bladder cancer cell invasion. Hipk2 knockdown resulted in decrease of E-cadherin expression and increase of N-cadherin and fibronectin expression, indicated that epithelial-mesenchymal transition (EMT) was induced. We further demonstrated that Hipk2 knockdown induced Wnt signaling activation and β-catenin nuclear localization. Finally, we confirmed that Hipk2 inhibition promoted EMT and subsequent cell invasion, at least in part by activating Wnt signaling. These data suggest an important role of Hipk2 in regulating metastasis of bladder cancer and implicate the potential application of Hipk2 in bladder cancer therapy.

  2. Homeodomain Interacting Protein Kinase 2 Regulates Postnatal Development of Enteric Dopaminergic Neurons and Glia via BMP Signaling

    PubMed Central

    Chalazonitis, Alcmène; Tang, Amy A.; Shang, Yulei; Pham, Tuan D.; Hsieh, Ivy; Setlik, Wanda; Gershon, Michael D.; Huang, Eric J.

    2011-01-01

    Trophic factor signaling is important for the migration, differentiation and survival of enteric neurons during development. The mechanisms that regulate the maturation of enteric neurons in postnatal life, however, are poorly understood. Here, we show that transcriptional cofactor HIPK2 (homeodomain interacting protein kinase 2) is required for the maturation of enteric neurons and for regulating gliogenesis during postnatal development. Mice lacking HIPK2 display a spectrum of gastrointestinal (GI) phenotypes, including distention of colon and slowed GI transit time. Although loss of HIPK2 does not affect enteric neuron in prenatal development, a progressive loss of enteric neurons occurs during postnatal life in Hipk2−/− -mutant mice that preferentially affects the dopaminergic population of neurons in the caudal region of the intestine. The mechanism by which HIPK2 regulates postnatal enteric neuron development appears to involve the response of enteric neurons to bone morphogenetic proteins (BMPs). Specifically, compared to wild type mice, a larger proportion of enteric neurons in Hipk2−/−mutants have abnormally high level of phosphorylated Smad1/5/8. Consistent with the ability of BMP signaling to promote gliogenesis, Hipk2−/− mutants show a significant increase in glia in the ENS. In addition, numbers of autophagosomes are increased in enteric neurons in Hipk2−/−mutants and synaptic maturation is arrested. These results reveal a new role for HIPK2 as an important transcriptional cofactor that regulates the BMP signaling pathway in the maintenance of enteric neurons and glia, and further suggest that HIPK2 and its associated signaling mechanisms may be therapeutically altered to promote postnatal neuronal maturation. PMID:21957238

  3. The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

    SciTech Connect

    Kong, Kyoung-Ah; Gadi, Jogeswar; Park, Hyoung Woo; Bok, Jinwoong Kim, Myoung Hee

    2008-12-05

    Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 {mu}M, and appeared to plateau above a concentration of 1 {mu}M. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.

  4. A role for the HOXB7 homeodomain protein in DNA repair.

    PubMed

    Rubin, Ethel; Wu, Xinyan; Zhu, Tao; Cheung, Joyce C Y; Chen, Hexin; Lorincz, Annaka; Pandita, Raj K; Sharma, Girdhar G; Ha, Hyo Chol; Gasson, Judith; Hanakahi, Les A; Pandita, Tej K; Sukumar, Saraswati

    2007-02-15

    Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo. Recent evidence suggests that the maintenance of specific HOX expression patterns is necessary for regulating the homeostasis of adult tissues as well. In this study, HOXB7 transformed human mammary epithelial cells, MCF10A, to grow in minimally supplemented medium, to form colonies in Matrigel, and display resistance to ionizing radiation. Searching for protein partners of HOXB7 that might contribute to resistance to ionizing radiation, we identified four HOXB7-binding proteins by GST pull-down/affinity chromatography and confirmed their interactions by coimmunoprecipitation in vivo. Interestingly, all four HOXB7-binding proteins shared functions as genomic caretakers and included members of the DNA-dependent protein kinase holoenzyme (Ku70, Ku80, DNA-PK(cs)) responsible for DNA double-strand break repair by nonhomologous end joining pathway and poly(ADP) ribose polymerase. Exogenous and endogenous expression of HOXB7 enhanced nonhomologous end joining and DNA repair functions in vitro and in vivo, which were reversed by silencing HOXB7. This is the first mechanistic study providing definitive evidence for the involvement of any HOX protein in DNA double-strand break repair.

  5. Runx2 Trans-Activation Mediated by the Msx2-Interacting Nuclear Target Requires Homeodomain Interacting Protein Kinase-3

    PubMed Central

    Sierra, Oscar L.; Towler, Dwight A.

    2010-01-01

    Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2 activation. Intriguingly, Runx1 cannot replace Runx2 in this assay. To better understand this Runx2 signaling cascade, we performed structure-function analysis of the Runx2-MINT trans-activation relationship. Systematic truncation and domain swapping in Runx1:Runx2 chimeras identified that the unique Runx2 activation domain 3 (AD3), encompassed by residues 316–421, conveys MINT+FGF2 trans-activation in transfection assays. Ala mutagenesis of Runx2 Ser/Thr residues identified that S301 and T326 in AD3 are necessary for full MINT+FGF2 trans-activation. Conversely, phosphomimetic Asp substitution of these AD3 Ser/Thr residues enhanced activation by MINT. Adjacent Pro residues implicated regulation by a proline-directed protein kinase (PDPK). Systematic screening with PDPK inhibitors identified that the casein kinase-2/homeodomain-interacting protein kinase (HIPK)/dual specificity tyrosine phosphorylation regulated kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), but not ERK, c-Jun N-terminal kinase, p38MAPK, or other casein kinase-2 inhibitors, abrogated Runx2-, MINT-, and FGF2-activation. Systematic small interfering RNA-mediated silencing of DMAT-inhibited PDPKs revealed that HIPK3 depletion reduced MINT+FGF2-dependent activation of Runx2. HIPK3 and Runx2 coprecipitate after in vitro transcription-translation, and recombinant HIPK3 recognizes Runx2 AD3 as kinase substrate. Furthermore, DMAT treatment and HIPK3 RNAi inhibited MINT+FGF2 activation of

  6. Homeodomain Protein Otp and Activity-Dependent Splicing Modulate Neuronal Adaptation to Stress

    PubMed Central

    Amir-Zilberstein, Liat; Blechman, Janna; Sztainberg, Yehezkel; Norton, William H.J.; Reuveny, Adriana; Borodovsky, Nataliya; Tahor, Maayan; Bonkowsky, Joshua L.; Bally-Cuif, Laure; Chen, Alon; Levkowitz, Gil

    2015-01-01

    SUMMARY Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal’s adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing. PMID:22284183

  7. Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.

    PubMed Central

    Knight, S A; Tamai, K T; Kosman, D J; Thiele, D J

    1994-01-01

    Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome XVI, caused a loss of copper resistance in strains which possessed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain. Measurement of intracellular copper levels of the wild-type and cup9-1 mutant demonstrated that total intracellular copper concentrations were unaffected by CUP9. CUP9 mRNA levels were, however, down regulated by copper when yeast cells were grown with glucose but not with lactate or glycerol-ethanol as the sole carbon source. This down regulation was independent of the copper metalloregulatory transcription factor ACE1. The DNA sequence of CUP9 predicts an open reading frame of 306 amino acids in which a 55-amino-acid sequence showed 47% identity with the homeobox domain of the human proto-oncogene PBX1, suggesting that CUP9 is a DNA-binding protein which regulates the expression of important copper homeostatic genes. Images PMID:7969120

  8. Regulation of retinal axon growth by secreted Vax1 homeodomain protein

    PubMed Central

    Kim, Namsuk; Min, Kwang Wook; Kang, Kyung Hwa; Lee, Eun Jung; Kim, Hyoung-Tai; Moon, Kyunghwan; Choi, Jiheon; Le, Dai; Lee, Sang-Hee; Kim, Jin Woo

    2014-01-01

    Retinal ganglion cell (RGC) axons of binocular animals cross the midline at the optic chiasm (OC) to grow toward their synaptic targets in the contralateral brain. Ventral anterior homeobox 1 (Vax1) plays an essential role in the development of the OC by regulating RGC axon growth in a non-cell autonomous manner. In this study, we identify an unexpected function of Vax1 that is secreted from ventral hypothalamic cells and diffuses to RGC axons, where it promotes axonal growth independent of its transcription factor activity. We demonstrate that Vax1 binds to extracellular sugar groups of the heparan sulfate proteoglycans (HSPGs) located in RGC axons. Both Vax1 binding to HSPGs and subsequent penetration into the axoplasm, where Vax1 activates local protein synthesis, are required for RGC axonal growth. Together, our findings demonstrate that Vax1 possesses a novel RGC axon growth factor activity that is critical for the development of the mammalian binocular visual system. DOI: http://dx.doi.org/10.7554/eLife.02671.001 PMID:25201875

  9. Control of sex-specific apoptosis in C. elegans by the BarH homeodomain protein CEH-30 and the transcriptional repressor UNC-37/Groucho.

    PubMed

    Peden, Erin; Kimberly, Elizabeth; Gengyo-Ando, Keiko; Mitani, Shohei; Xue, Ding

    2007-12-01

    Apoptosis is essential for proper development and tissue homeostasis in metazoans. It plays a critical role in generating sexual dimorphism by eliminating structures that are not needed in a specific sex. The molecular mechanisms that regulate sexually dimorphic apoptosis are poorly understood. Here we report the identification of the ceh-30 gene as a key regulator of sex-specific apoptosis in Caenorhabditis elegans. Loss-of-function mutations in ceh-30 cause the ectopic death of male-specific CEM neurons. ceh-30 encodes a BarH homeodomain protein that acts downstream from the terminal sex determination gene tra-1, but upstream of, or in parallel to, the cell-death-initiating gene egl-1 to protect CEM neurons from undergoing apoptosis in males. The second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A and a POU-type homeodomain protein UNC-86 and acts as a sensor to regulate proper specification of the CEM cell fate. Surprisingly, the N terminus of CEH-30 but not its homeodomain is critical for CEH-30's cell death inhibitory activity in CEMs and contains a conserved eh1/FIL domain that is important for the recruitment of the general transcriptional repressor UNC-37/Groucho. Our study suggests that ceh-30 defines a critical checkpoint that integrates the sex determination signal TRA-1 and the cell fate determination and survival signal UNC-86 to control the sex-specific activation of the cell death program in CEMs through the general transcription repressor UNC-37.

  10. Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins

    PubMed Central

    Mead, Paul E.; Zhou, Yi; Lustig, Kevin D.; Huber, Tara L.; Kirschner, Marc W.; Zon, Leonard I.

    1998-01-01

    We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expressed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation. PMID:9736722

  11. Heterodimeric Pbx-Prep1 homeodomain protein binding to the glucagon gene restricting transcription in a cell type-dependent manner.

    PubMed

    Herzig, S; Fuzesi, L; Knepel, W

    2000-09-08

    Homeodomain proteins specify developmental pathways and cell-specific gene transcription whereby proteins of the PBC subclass can direct target gene specificity of Hox proteins. Proteins encoded by nonclustered homeobox genes have been shown to be essential for cell lineage differentiation and gene expression in pancreatic islets. Using specific antiserum in an electrophoretic mobility shift assay and in vitro transcribed/translated proteins, the nuclear proteins binding domain B of the G3 enhancer-like element of the glucagon gene were identified in the present study as heterodimers consisting of the ubiquitously expressed homeodomain protein Prep1 and the also widely expressed PBC homeoprotein Pbx (isoform 1a, 1b, or 2). These heterodimeric complexes were found to bind also to the glucagon cAMP response element and to a newly identified element termed G5 (from -169 to -140). Whereas the expression of Prep1 or Pbx forms alone had no effect, coexpression of Pbx1a/1b-Prep1 inhibited the glucagon promoter when activated by cotransfected Pax6 or another transcription factor in non-glucagon-producing cells. In contrast, in glucagon-producing pancreatic islet cells, Pbx-Prep1 had no effect on GAL4-Pax6-induced mutant glucagon promoter activity or on Pax6-dependent wild-type glucagon promoter activity. Furthermore, 5'-deletion of G5 enhanced glucagon promoter activity in a non-glucagon-producing cell line but not in glucagon-producing islet cells. This study thus identifies a novel target and Hox-independent function of Pbx-Prep1 heterodimers that, through repression of glucagon gene transcription in non-glucagon-producing cells, may help to establish islet cell-specific expression of the glucagon gene.

  12. The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila

    PubMed Central

    Roignant, Jean-Yves; Legent, Kevin; Janody, Florence; Treisman, Jessica E.

    2010-01-01

    Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. We have found that the transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. We show that two LIM-homeodomain proteins, Arrowhead and Lim1, are expressed in the region of the eye-antennal disc affected in Chip mutants, and that both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue. PMID:20040493

  13. A Structural Basis for the Regulation of the LIM-Homeodomain Protein Islet 1 (Isl1) by Intra- and Intermolecular Interactions*

    PubMed Central

    Gadd, Morgan S.; Jacques, David A.; Nisevic, Ivan; Craig, Vanessa J.; Kwan, Ann H.; Guss, J. Mitchell; Matthews, Jacqueline M.

    2013-01-01

    Islet 1 (Isl1) is a transcription factor of the LIM-homeodomain (LIM-HD) protein family and is essential for many developmental processes. LIM-HD proteins all contain two protein-interacting LIM domains, a DNA-binding homeodomain (HD), and a C-terminal region. In Isl1, the C-terminal region also contains the LIM homeobox 3 (Lhx3)-binding domain (LBD), which interacts with the LIM domains of Lhx3. The LIM domains of Isl1 have been implicated in inhibition of DNA binding potentially through an intramolecular interaction with or close to the HD. Here we investigate the LBD as a candidate intramolecular interaction domain. Competitive yeast-two hybrid experiments indicate that the LIM domains and LBD from Isl1 can interact with apparently low affinity, consistent with no detection of an intermolecular interaction in the same system. Nuclear magnetic resonance studies show that the interaction is specific, whereas substitution of the LBD with peptides of the same amino acid composition but different sequence is not specific. We solved the crystal structure of a similar but higher affinity complex between the LIM domains of Isl1 and the LIM interaction domain from the LIM-HD cofactor protein LIM domain-binding protein 1 (Ldb1) and used these coordinates to generate a homology model of the intramolecular interaction that indicates poorer complementarity for the weak intramolecular interaction. The intramolecular interaction in Isl1 may provide protection against aggregation, minimize unproductive DNA binding, and facilitate cofactor exchange within the cell. PMID:23750000

  14. Regulation of proliferation in developing human tooth germs by MSX muscle segment homeodomain proteins and cyclin-dependent kinase inhibitor p19(INK4d).

    PubMed

    Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Brakus, Snjezana Mardesic; Saraga-Babic, Mirna

    2017-09-21

    Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19(INK4d). p19(INK4d) induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19(INK4d) by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19(INK4d) in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19(INK4d) throughout the investigated period indicates that p19(INK4d) plays active role during human tooth development. Furthermore, comparison of expression domains of p19(INK4d) with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

  15. A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    PubMed Central

    Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S.; Jiang, Cai-Zhong

    2014-01-01

    Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence. PMID:24551088

  16. Phage phi29 DNA replication organizer membrane protein p16.7 contains a coiled coil and a dimeric, homeodomain-related, functional domain.

    PubMed

    Muñoz-Espín, Daniel; Mateu, Mauricio G; Villar, Laurentino; Marina, Anabel; Salas, Margarita; Meijer, Wilfried J J

    2004-11-26

    The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication. Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization. A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the varphi29 terminal protein. Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil. However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization. This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein. Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins. Based on the results, a structural model of p16.7 is presented.

  17. Cyclin E-CDK2 protein phosphorylates plant homeodomain finger protein 8 (PHF8) and regulates its function in the cell cycle.

    PubMed

    Sun, Liping; Huang, Yan; Wei, Qian; Tong, Xiaomei; Cai, Rong; Nalepa, Grzegorz; Ye, Xin

    2015-02-13

    Cyclin E-CDK2 is a key regulator in G1/S transition. Previously, we identified a number of CDK2-interacting proteins, including PHF8 (plant homeodomain finger protein 8). In this report, we confirmed that PHF8 is a novel cyclin E-CDK2 substrate. By taking the approach of mass spectrometry, we identified that PHF8 Ser-844 is phosphorylated by cyclin E-CDK2. Immunoblotting analysis indicated that WT PHF8 demethylates histone H3K9me2 more efficiently than the cyclin E-CDK2 phosphorylation-deficient PHF8-S844A mutant. Furthermore, flow cytometry analysis showed that WT PHF8 promotes S phase progression more robustly than PHF8-S844A. Real-time PCR results demonstrated that PHF8 increases transcription of cyclin E, E2F3, and E2F7 to significantly higher levels compared with PHF8-S844A. Further analysis by ChIP assay indicated that PHF8 binds to the cyclin E promoter stronger than PHF8-S844A and reduces the H3K9me2 level at the cyclin E promoter more efficiently than PHF8-S844A. In addition, we found that cyclin E-CDK2-mediated phosphorylation of PHF8 Ser-844 promotes PHF8-dependent rRNA transcription in luciferase reporter assays and real-time PCR. Taken together, these results indicate that cyclin E-CDK2 phosphorylates PHF8 to stimulate its demethylase activity to promote rRNA transcription and cell cycle progression.

  18. The Arabidopsis Zinc Finger-Homeodomain Genes Encode Proteins with Unique Biochemical Properties That Are Coordinately Expressed during Floral Development1

    PubMed Central

    Tan, Queenie K.-G.; Irish, Vivian F.

    2006-01-01

    Arabidopsis (Arabidopsis thaliana) contains approximately 100 homeobox genes, many of which have been shown to play critical roles in various developmental processes. Here we characterize the zinc finger-homeodomain (ZF-HD) subfamily of homeobox genes, consisting of 14 members in Arabidopsis. We demonstrate that the HDs of the ZF-HD proteins share some similarities with other known HDs in Arabidopsis, but they contain distinct features that cluster them as a unique class of plant HD-containing proteins. We have carried out mutational analyses to show that the noncanonical residues present in the HDs of this family of proteins are important for function. Yeast (Saccharomyces cerevisiae) two-hybrid matrix analyses of the ZF-HD proteins reveal that these proteins both homo- and heterodimerize, which may contribute to greater selectivity in DNA binding. These assays also show that most of these proteins do not contain an intrinsic activation domain, suggesting that interactions with other factors are required for transcriptional activation. We also show that the family members are all expressed predominantly or exclusively in floral tissue, indicating a likely regulatory role during floral development. Furthermore, we have identified loss-of-function mutations for six of these genes that individually show no obvious phenotype, supporting the idea that the encoded proteins have common roles in floral development. Based on these results, we propose the ZF-HD gene family encodes a group of transcriptional regulators with unique biochemical activities that play overlapping regulatory roles in Arabidopsis floral development. PMID:16428600

  19. Aberrant development of the suprachiasmatic nucleus and circadian rhythms in mice lacking the homeodomain protein Six6.

    PubMed

    Clark, Daniel D; Gorman, Michael R; Hatori, Megumi; Meadows, Jason D; Panda, Satchidananda; Mellon, Pamela L

    2013-02-01

    The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and β-galactosidase, we demonstrated the lack of these SCN markers in all Six6-null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN.

  20. The LIM homeodomain protein Lhx6 regulates maturation of interneurons and network excitability in the mammalian cortex.

    PubMed

    Neves, Guilherme; Shah, Mala M; Liodis, Petros; Achimastou, Angeliki; Denaxa, Myrto; Roalfe, Grant; Sesay, Abdul; Walker, Matthew C; Pachnis, Vassilis

    2013-08-01

    Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst(+) interneurons, but relatively minor and localized changes in Pva(+) interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst(+) interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

  1. Aberrant Development of the Suprachiasmatic Nucleus and Circadian Rhythms in Mice Lacking the Homeodomain Protein Six6

    PubMed Central

    Clark, Daniel D.; Gorman, Michael R.; Hatori, Megumi; Meadows, Jason D.; Panda, Satchidananda; Mellon, Pamela L.

    2013-01-01

    The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and β-galactosidase, we demonstrated the lack of these SCN markers in all Six6- null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN. PMID:23382588

  2. Targeted disruption of the murine homeodomain-interacting protein kinase-2 causes growth deficiency in vivo and cell cycle arrest in vitro.

    PubMed

    Trapasso, Francesco; Aqeilan, Rami I; Iuliano, Rodolfo; Visone, Rosa; Gaudio, Eugenio; Ciuffini, Laura; Alder, Hansjuerg; Paduano, Francesco; Pierantoni, Giovanna Maria; Soddu, Silvia; Croce, Carlo M; Fusco, Alfredo

    2009-04-01

    The homeodomain-interacting protein kinase 2 (HIPK2) protein is a member of a recently identified family of nuclear protein kinases that are well conserved in various organisms. HIPK2 can bind to several homeotic factors and to a series of proteins involved in the regulation of cell survival and proliferation in response to morphogenetic and genotoxic signals. Here we report Hipk2-targeted disruption in mouse; Hipk2(-/-) mice are viable and fertile but significantly smaller than their wild-type littermates. This feature is present at birth and retained throughout the mouse adulthood. Mouse embryo fibroblasts from Hipk2(-/-) mice show a reduced proliferation rate, compared to the wild-type counterparts, with accumulation in the G0/G1 phase of the cell cycle and altered levels of the cell cycle regulators cyclin D and CDK6. Restoration of wild-type HIPK2 expression in Hipk2(-/-) cells rescues the normal phenotype supporting a role for HIPK2 in the regulation of cell proliferation.

  3. NMR solution structure of the bicoid homeodomain bound to DNA and molecular dynamics simulations of the homeodomain/DNA complex

    NASA Astrophysics Data System (ADS)

    Baird-Titus, Jamie M.

    The homeodomain is a common DNA recognition motif consisting of three helices and an N-terminal arm that serves as a valuable model for exploring the basis of specific DNA recognition by proteins. Recognition of specific DNA sites, loosely defined by a TAAT core, is dependent on the side-chains of key amino acids in the N-terminal arm and the third "recognition" helix of the homeodomain. While much is known about homeodomain/DNA recognition, key questions concerning the role of individual amino acids and the extent of side-chain, DNA, and water dynamics during recognition remain, often focusing on the dynamic role of position 50 during recognition of the two bases immediately 3' to the 5'-TAAT-3'/3'-ATTA-5' core (ATTANN). The Bicoid homeodomain provides an interesting model system for addressing these and other questions, serving as the only known homeodomain that has a dual role in both transcriptional (DNA-binding) and translational (RNA-binding) control, discriminating between these two functions by a single amino acid, arginine 54. To add to the understanding of both general protein/DNA recognition and to the specific function of the Bicoid transcription factor homeodomain, we have determined the solution structure of the Bicoid homeodomain bound to the consensus duplex B-DNA binding site 5'-TAATCC-3'/3'-ATTAGG-5'. Our structure indicates that the Bicoid homeodomain exhibits variation from other homeodomain structures at the end of helix I, and NMR resonance line broadening of the K50 and R54 side-chains, consistent with side-chain motion and supportive of the adaptive-recognition theory of protein/DNA interactions.

  4. The HMX homeodomain protein MLS-2 regulates cleavage orientation, cell proliferation and cell fate specification in the C. elegans postembryonic mesoderm.

    PubMed

    Jiang, Yuan; Horner, Vanessa; Liu, Jun

    2005-09-01

    The proper formation of a complex multicellular organism requires the precise coordination of many cellular events, including cell proliferation, cell fate specification and differentiation. The C. elegans postembryonic mesodermal lineage, the M lineage, allows us to study mechanisms coordinating these events at single cell resolution. We have identified an HMX homeodomain protein MLS-2 in a screen for factors required for M lineage patterning. The MLS-2 protein is present in nuclei of undifferentiated cells in the early M lineage and in a subset of head neurons. In the M lineage, MLS-2 activity appears to be tightly regulated at the fourth round of cell division, coincident with the transition from proliferation to differentiation. A predicted null allele of mls-2, cc615, causes reduced cell proliferation in the M lineage, whereas a semi-dominant, gain-of-function allele, tm252, results in increased cell proliferation. Loss or overexpression of mls-2 also affects cleavage orientation and cell fate specification in the M lineage. We show that the increased cell proliferation in mls-2(tm252) mutants requires CYE-1, a G1 cell cycle regulator. Furthermore, the C. elegans Myod homolog HLH-1 acts downstream of mls-2 to specify M-derived coelomocyte cell fates. Thus MLS-2 functions in a cell type-specific manner to regulate both cell proliferation and cell fate specification.

  5. The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage.

    PubMed

    Reuven, Nina; Adler, Julia; Porat, Ziv; Polonio-Vallon, Tilman; Hofmann, Thomas G; Shaul, Yosef

    2015-07-03

    The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type

    PubMed Central

    Choi, Seong-Kyoon; Huh, Yang Hoon; Fang, Zi; Park, Seo Jin; Kim, Myoung Ok; Ryoo, Zae Young; Kang, Kyeongjin; Kweon, Hee-Seok; Jeon, Won Bae; Li, Chris; Kim, Kyuhyung

    2015-01-01

    The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes. PMID:26305787

  7. The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type.

    PubMed

    Kim, Jinmahn; Yeon, Jihye; Choi, Seong-Kyoon; Huh, Yang Hoon; Fang, Zi; Park, Seo Jin; Kim, Myoung Ok; Ryoo, Zae Young; Kang, Kyeongjin; Kweon, Hee-Seok; Jeon, Won Bae; Li, Chris; Kim, Kyuhyung

    2015-08-01

    The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

  8. The HMX/NKX homeodomain protein MLS-2 specifies the identity of the AWC sensory neuron type via regulation of the ceh-36 Otx gene in C. elegans

    PubMed Central

    Kim, Kyuhyung; Kim, Rinho; Sengupta, Piali

    2010-01-01

    The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. Here, we show that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. We also show that MLS-2 acts in additional neuron types to regulate their development and differentiation. Our analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system. PMID:20150279

  9. The Nkx5/HMX homeodomain protein MLS-2 is required for proper tube cell shape in the C.elegans excretory system

    PubMed Central

    Abdus-Saboor, Ishmail; Stone, Craig E.; Murray, John I.; Sundaram, Meera V.

    2012-01-01

    Cells perform wide varieties of functions that are facilitated, in part, by adopting unique shapes. Many of the genes and pathways that promote cell fate specification have been elucidated. However, relatively few transcription factors have been identified that promote shape acquisition after fate specification. Here we show that the Nkx5/HMX homeodomain protein MLS-2 is required for cellular elongation and shape maintenance of two tubular epithelial cells in the C.elegans excretory system, the duct and pore cells. The Nkx5/HMX family is highly conserved from sea urchins to humans, with known roles in neuronal and glial development. MLS-2 is expressed in the duct and pore, and defects in mls-2 mutants first arise when the duct and pore normally adopt unique shapes. MLS-2 cooperates with the EGF-Ras-ERK pathway to turn on the LIN-48/Ovo transcription factor in the duct cell during morphogenesis. These results reveal a novel interaction between the Nkx5/HMX family and the EGF-Ras pathway and implicate a transcription factor, MLS-2, as a regulator of cell shape. PMID:22537498

  10. The Nkx5/HMX homeodomain protein MLS-2 is required for proper tube cell shape in the C. elegans excretory system.

    PubMed

    Abdus-Saboor, Ishmail; Stone, Craig E; Murray, John I; Sundaram, Meera V

    2012-06-15

    Cells perform wide varieties of functions that are facilitated, in part, by adopting unique shapes. Many of the genes and pathways that promote cell fate specification have been elucidated. However, relatively few transcription factors have been identified that promote shape acquisition after fate specification. Here we show that the Nkx5/HMX homeodomain protein MLS-2 is required for cellular elongation and shape maintenance of two tubular epithelial cells in the C. elegans excretory system, the duct and pore cells. The Nkx5/HMX family is highly conserved from sea urchins to humans, with known roles in neuronal and glial development. MLS-2 is expressed in the duct and pore, and defects in mls-2 mutants first arise when the duct and pore normally adopt unique shapes. MLS-2 cooperates with the EGF-Ras-ERK pathway to turn on the LIN-48/Ovo transcription factor in the duct cell during morphogenesis. These results reveal a novel interaction between the Nkx5/HMX family and the EGF-Ras pathway and implicate a transcription factor, MLS-2, as a regulator of cell shape. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. The HMX/NKX homeodomain protein MLS-2 specifies the identity of the AWC sensory neuron type via regulation of the ceh-36 Otx gene in C. elegans.

    PubMed

    Kim, Kyuhyung; Kim, Rinho; Sengupta, Piali

    2010-03-01

    The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. Here, we show that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. We also show that MLS-2 acts in additional neuron types to regulate their development and differentiation. Our analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system.

  12. LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum

    PubMed Central

    Zhao, Yangu; Kwan, Kin-Ming; Mailloux, Christina M.; Lee, Woon-Kyu; Grinberg, Alexander; Wurst, Wolfgang; Behringer, Richard R.; Westphal, Heiner

    2007-01-01

    Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum. PMID:17664423

  13. CFL1, a WW domain protein, regulates cuticle development by modulating the function of HDG1, a class IV homeodomain transcription factor, in rice and Arabidopsis.

    PubMed

    Wu, Renhong; Li, Shibai; He, Shan; Wassmann, Friedrich; Yu, Caihong; Qin, Genji; Schreiber, Lukas; Qu, Li-Jia; Gu, Hongya

    2011-09-01

    Plants have a chemically heterogeneous lipophilic layer, the cuticle, which protects them from biotic and abiotic stresses. The mechanisms that regulate cuticle development are poorly understood. We identified a rice (Oryza sativa) dominant curly leaf mutant, curly flag leaf1 (cfl1), and cloned CFL1, which encodes a WW domain protein. We overexpressed both rice and Arabidopsis CFL1 in Arabidopsis thaliana; these transgenic plants showed severely impaired cuticle development, similar to that in cfl1 rice. Reduced expression of At CFL1 resulted in reinforcement of cuticle structure. At CFL1 was predominantly expressed in specialized epidermal cells and in regions where dehiscence and abscission occur. Biochemical evidence showed that At CFL1 interacts with HDG1, a class IV homeodomain-leucine zipper transcription factor. Suppression of HDG1 function resulted in similar defective cuticle phenotypes in wild-type Arabidopsis but much alleviated phenotypes in At cfl1-1 mutants. The expression of two cuticle development-associated genes, BDG and FDH, was downregulated in At CFL1 overexpressor and HDG1 suppression plants. HDG1 binds to the cis-element L1 box, which exists in the regulatory regions of BDG and FDH. Our results suggest that rice and Arabidopsis CFL1 negatively regulate cuticle development by affecting the function of HDG1, which regulates the downstream genes BDG and FDH.

  14. Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper protein, regulates leaf growth by promoting cell expansion and endoreduplication.

    PubMed

    Hur, Yoon-Sun; Um, Ji-Hyun; Kim, Sunghan; Kim, Kyunga; Park, Hee-Jung; Lim, Jong-Seok; Kim, Woo-Young; Jun, Sang Eun; Yoon, Eun Kyung; Lim, Jun; Ohme-Takagi, Masaru; Kim, Donggiun; Park, Jongbum; Kim, Gyung-Tae; Cheon, Choong-Ill

    2015-01-01

    Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper class I (HD-Zip I) gene, is highly expressed in leaves and stems, and induced by abiotic stresses, but its role in development remains obscure. To understand its function during plant development, we studied the effects of loss and gain of function. Expression of ATHB12 fused to the EAR-motif repression domain (SRDX) - P35 S ::ATHB12SRDX (A12SRDX) and PATHB 12 ::ATHB12SRDX - slowed both leaf and root growth, while the growth of ATHB12-overexpressing seedlings (A12OX) was accelerated. Microscopic examination revealed changes in the size and number of leaf cells. Ploidy was reduced in A12SRDX plants, accompanied by decreased cell expansion and increased cell numbers. By contrast, cell size was increased in A12OX plants, along with increased ploidy and elevated expression of cell cycle switch 52s (CCS52s), which are positive regulators of endoreduplication, indicating that ATHB12 promotes leaf cell expansion and endoreduplication. Overexpression of ATHB12 led to decreased phosphorylation of Arabidopsis thaliana ribosomal protein S6 (AtRPS6), a regulator of cell growth. In addition, induction of ATHB12 in the presence of cycloheximide increased the expression of several genes related to cell expansion, such as EXPANSIN A10 (EXPA10) and DWARF4 (DWF4). Our findings strongly suggest that ATHB12 acts as a positive regulator of endoreduplication and cell growth during leaf development.

  15. The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1

    SciTech Connect

    Plant, Kathryn E.; Anderson, Elizabeth; Simecek, Nicole; Brown, Richard; Forster, Sam; Spinks, Jenny; Toms, Nick; Gibson, G. Gordon; Lyon, Jon; Plant, Nick

    2009-02-15

    The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism.

  16. Homeodomains, Hedgehogs, and Happiness.

    PubMed

    Scott, Matthew P

    2016-01-01

    Developmental biologists have had a spectacular quarter century of discoveries, building on many decades of work earlier, discovering molecular, cellular, and genetic mechanisms that underlie the magical process by which an egg becomes a plant or animal. Among the discoveries were homeodomains, DNA-binding domains that allow transcription factors to recognize their target genes, and the Hedgehog signaling pathway, which is used in many organs and tissues for communication among cells. The experience of unveiling the mechanisms and molecules connected to both of these findings has been remarkable, joyful, difficult, and a time of great teamwork and collaboration within and between laboratory groups. More than ever it is possible to discern the evolutionary processes, and their mechanisms, that led to the diversity of life on earth. A huge amount of work remains to be done to obtain a broad understanding of what happened and how development works.

  17. The homeodomain proteins PBX and MEIS1 are accessory factors that enhance thyroid hormone regulation of the malic enzyme gene in hepatocytes.

    PubMed

    Wang, Y; Yin, L; Hillgartner, F B

    2001-06-29

    Triiodothyronine (T3) stimulates a robust increase (>40-fold) in transcription of the malic enzyme gene in chick embryo hepatocytes. Previous work has shown that optimal T3 regulation of malic enzyme transcription is dependent on the presence of an accessory element (designated as region E) that immediately flanks a cluster of five T3 response elements in the malic enzyme gene. Here, we have analyzed the binding of nuclear proteins to region E and investigated the mechanism by which region E enhances T3 responsiveness. In nuclear extracts from hepatocytes, region E binds heterodimeric complexes consisting of the homeodomain proteins PBX and MEIS1. Region E contains four consecutive PBX/MEIS1 half-sites. PBX-MEIS1 heterodimers bind the first and second half-sites, the third and fourth half-sites, and the first and fourth half-sites. The configuration conferring the greatest increase in T3 responsiveness consists of the first and fourth half-sites that are separated by 7 nucleotides. Stimulation of T3 response element functions by region E does not require the presence of additional malic enzyme sequences. In pull-down experiments, PBX1a and PBX1b specifically bind the nuclear T3 receptor-alpha, and this interaction is enhanced by the presence of T3. A T3 receptor-alpha region containing the DNA binding domain plus flanking sequences (amino acids 21-157) is necessary and sufficient for binding to PBX1a and PBX1b. These results indicate that PBX-MEIS1 complexes interact with nuclear T3 receptors to enhance T3 regulation of malic enzyme transcription in hepatocytes.

  18. CaHDZ27, a Homeodomain-Leucine Zipper I (HD-Zip I) Protein, Positively Regulates the Resistance to Ralstonia solanacearum Infection in Pepper.

    PubMed

    Mou, Shaoliang; Liu, Zhiqin; Gao, Feng; Yang, Sheng; Su, Meixia; Shen, Lei; Wu, Yang; He, Shuilin

    2017-08-25

    Homeodomain-leucine zipper class I (HD-Zip I) transcription factors (TFs) have been functionally characterized in plant responses to abiotic stresses, but their roles in plant immunity are poorly understood. Here, a HD-Zip I gene, CaHZ27, was isolated from pepper (Capsicum annum) and characterized for its role in pepper immunity. Quantitative real-time PCR showed that CaHDZ27 was transcriptionally induced by Ralstonia solanacearum inoculation and exogenous application of methyl jasmonate (MeJA), salicylic acid (SA), or ethephon (ETH). The CaHDZ27-GFP (green fluorescent protein) fused protein was targeted exclusively to the nucleus. Chromatin immunoprecipitation (ChIP) demonstrated that CaHDZ27 bound to the 9-bp pseudopalindromic element (CAATAATTG) and triggered GUS expression in a CAATAATTG-dependent manner. Virus-induced gene silencing (VIGS) of CaHDZ27 significantly attenuated the resistance of pepper plants against R. solanacearum and downregulated defense-related marker genes including CaHIR1, CaACO1, CaPR1, CaPR4, CaPO2, and CaBPR1. By contrast, transient overexpression of CaHDZ27 triggered strong cell death mediated by the hypersensitive response (HR), and upregulated the tested immunity-associated marker genes. Ectopic CaHDZ27 expression in tobacco enhances its resistance against R. solanacearum. These results collectively suggest that CaHDZ27 functions as a positive regulator in pepper resistance against R. solanacearum. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays indicate that CaHDZ27 monomers bind with each other, and this binding is enhanced significantly by R. solanacearum inoculation. We speculate that homodimerization of CaHZ27 might play a role in pepper response to R. solanacearum, further direct evidence is required to confirm it.

  19. Genetic ablation of homeodomain-interacting protein kinase 2 selectively induces apoptosis of cerebellar Purkinje cells during adulthood and generates an ataxic-like phenotype

    PubMed Central

    Anzilotti, S; Tornincasa, M; Gerlini, R; Conte, A; Brancaccio, P; Cuomo, O; Bianco, G; Fusco, A; Annunziato, L; Pignataro, G; Pierantoni, G M

    2015-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented coregulator of an increasing number of transcription factors and cofactors involved in cell death and proliferation in several organs and systems. As Hipk2−/− mice show behavioral abnormalities consistent with cerebellar dysfunction, we investigated whether Hipk2 is involved in these neurological symptoms. To this aim, we characterized the postnatal developmental expression profile of Hipk2 in the brain cortex, hippocampus, striatum, and cerebellum of mice by real-time PCR, western blot analysis, and immunohistochemistry. Notably, we found that whereas in the brain cortex, hippocampus, and striatum, HIPK2 expression progressively decreased with age, that is, from postnatal day 1 to adulthood, it increased in the cerebellum. Interestingly, mice lacking Hipk2 displayed atrophic lobules and a visibly smaller cerebellum than did wild-type mice. More important, the cerebellum of Hipk2−/− mice showed a strong reduction in cerebellar Purkinje neurons during adulthood. Such reduction is due to the activation of an apoptotic process associated with a compromised proteasomal function followed by an unpredicted accumulation of ubiquitinated proteins. In particular, Purkinje cell dysfunction was characterized by a strong accumulation of ubiquitinated β-catenin. Moreover, our behavioral tests showed that Hipk2−/− mice displayed muscle and balance impairment, indicative of Hipk2 involvement in cerebellar function. Taken together, these results indicate that Hipk2 exerts a relevant role in the survival of cerebellar Purkinje cells and that Hipk2 genetic ablation generates cerebellar dysfunction compatible with an ataxic-like phenotype. PMID:26633710

  20. Homeodomain-interacting Protein Kinase-2 (HIPK2) Phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and Modulates Its DNA Binding Affinity

    PubMed Central

    Zhang, Qingchun; Wang, Yinsheng

    2008-01-01

    The chromosomal high-mobility group A (HMGA) proteins, comprising of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line ε promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. Synopsis Here we report that both HIPK2 and cdc2 phosphorylate HMGA1a at Ser-35, Thr-52 and Thr-77, but the two kinases exhibit different site preferences. Moreover, we found that HIPK2-induced phosphorylation of HMGA1a reduced the binding affinity of HMGA1a to DNA, and the drop in binding affinity was lower than that introduced by cdc2 phosphorylation, confirming

  1. Conformational changes in the PBX homeodomain and C-terminal extension upon binding DNA and HOX-derived YPWM peptides.

    PubMed

    Sprules, T; Green, N; Featherstone, M; Gehring, K

    2000-08-15

    PBX is a member of the three amino acid loop extension (TALE) class of homeodomains. PBX binds DNA cooperatively with HOX homeodomain proteins that contain a conserved YPWM motif. The amino acids immediately C-terminal to the PBX homeodomain increase the affinity of the homeodomain for its DNA site and HOX proteins. We have determined the structure of the free PBX homeodomain using NMR spectroscopy. Both the PBX homeodomain and the extended PBX homeodomain make identical contacts with a 5'-TGAT-3' DNA site and a YPWM peptide. A fourth alpha-helix, which forms upon binding to DNA, stabilizes the extended PBX structure. Variations in DNA sequence selectivity of heterodimeric PBX-HOX complexes depend on the HOX partner; however, a comparison of five different HOX-derived YPWM peptides showed that each bound to PBX in the same way, differing only in the strength of the association.

  2. Homeodomain interacting protein kinase 2 activation compromises endothelial cell response to laminar flow: protective role of p21(waf1,cip1,sdi1).

    PubMed

    Mattiussi, Stefania; Lazzari, Chiara; Truffa, Silvia; Antonini, Annalisa; Soddu, Silvia; Capogrossi, Maurizio C; Gaetano, Carlo

    2009-08-11

    In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear. To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 microg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm(2) did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function. This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.

  3. Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

    SciTech Connect

    Moon, Sunjin; Lee, Yong Woo; Kim, Woo Taek; Lee, Weontae

    2014-01-10

    Highlights: •We have determined solution structures of CEH-37 homedomain. •CEH-37 HD has a compact α-helical structure with HTH DNA binding motif. •Solution structure of CEH-37 HD shares its molecular topology with that of the homeodomain proteins. •Residues in the N-terminal region and HTH motif are important in binding to Caenorhabditis elegans telomeric DNA. •CEH-37 could play an important role in telomere function via DNA binding. -- Abstract: The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.

  4. Tumor suppressor von Hippel-Lindau (VHL) stabilization of Jade-1 protein occurs through plant homeodomains and is VHL mutation dependent.

    PubMed

    Zhou, Mina I; Wang, Hongmei; Foy, Rebecca L; Ross, Jonathan J; Cohen, Herbert T

    2004-02-15

    The von Hippel-Lindau (VHL) gene is the major renal cancer gene in adults. The mechanism of renal tumor suppression by VHL protein is only partly elucidated. VHL loss increases expression of the hypoxia-inducible factor alpha transcription factors. However, clinical and biochemical data indicate that the hypoxia-inducible factors are necessary but not sufficient for renal tumorigenesis, which suggests other VHL effector pathways are involved. Jade-1 protein interacts strongly with VHL and is most highly expressed in renal proximal tubules, precursor cells of renal cancer. Short-lived Jade-1 protein contains plant homeodomain (PHD) and candidate PEST degradation motifs and is substantially stabilized by VHL. The effect of VHL on Jade-1 protein abundance and relative protein stability was further examined in immunoblots and metabolic labeling experiments using two time points. VHL-Jade-1 binding was tested in coimmunoprecipitations. In cotransfection studies with wild-type VHL, the Jade-1 PHD-extended PHD module, not the candidate PEST domain, was required for full VHL-mediated stabilization. This module is also found in leukemia transcription factors AF10 and AF17, as well as closely related Jade-like proteins, which suggests all might be VHL regulated. Intriguingly, naturally occurring truncations and mutations of VHL affected wild-type Jade-1 binding and stabilization. Although the VHL beta domain was sufficient for Jade-1 binding, both the alpha and beta domains were required for Jade-1 stabilization. Thus, truncating VHL mutations, which are severe and associated with renal cancer development, prevented Jade-1 stabilization. Moreover, well-controlled cotransfection and metabolic labeling experiments revealed that VHL missense mutations that cause VHL disease without renal cancer, such as Tyr98His and Tyr112His, stabilized Jade-1 fully. In contrast, like the VHL truncations, VHL missense mutations commonly associated with renal cancer, such as Leu118Pro or Arg167

  5. Recognition rules for binding of homeodomains to operator DNA.

    PubMed

    Chirgadze, Yu N; Sivozhelezov, V S; Polozov, R V; Stepanenko, V A; Ivanov, V V

    2012-01-01

    The spatial arrangement of interfaces between homeodomain transcription factors and operator DNA has been considered. We analyzed the binding contacts for a representative set of 22 complexes of homeodomain transcription factors with a double-stranded operator DNA in the region of the major groove. It was shown that the recognition of DNA by the recognizing _-helix of protein is governed by two contact groups. Invariant protein-DNA group of contacts includes six contacts, formed by atomic groups of coding and non-coding DNA chains with the groups of amino acids. The recognizing _-helix forms contacts by polar groups of residues Trp2 (NE1), Asn5, and Lys9 with the canonical sequence T(1)A(2)A(3)T(4) of the coding DNA chain, and contacts by residues Lys0, Arg7 and Lys11 with the sequence A(4)X(5)X(6)X(7) of a non-coding DNA chain, where X is any nucleotide. Variable protein-DNA group of contacts comprises two groups bound with the sequence T(3)A(4)X(5)X(6) of the non- coding DNA-chain. These contacts are mainly with the bases and specify the binding pattern of individual homeodomains. The invariant contact group represents a recognition pattern for transcription factors of the homeodomain family: multiple adenine-asparagine contact and six position-specific phosphate contacts mainly with lysine or arginine. Within this group, we have found three most significant invariant contacts which allow deducing the recognition rules for homeodomains. These rules are inherent for different taxonomic groups of the homeodomain family and can distinguishing members of this family from any other family of transcription factors.

  6. Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans.

    PubMed

    Moon, Sunjin; Lee, Yong Woo; Kim, Woo Taek; Lee, Weontae

    2014-01-10

    The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.

  7. Phosphorylation of Krüppel-like Factor 3 (KLF3/BKLF) and C-terminal Binding Protein 2 (CtBP2) by Homeodomain-interacting Protein Kinase 2 (HIPK2) Modulates KLF3 DNA Binding and Activity*

    PubMed Central

    Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R.; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J.; Quinlan, Kate G. R.; Cordwell, Stuart J.; Funnell, Alister P. W.; Pearson, Richard C. M; Crossley, Merlin

    2015-01-01

    Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. PMID:25659434

  8. Paired-like homeodomain proteins Phox2a/Arix and Phox2b/NBPhox have similar genetic organization and independently regulate dopamine beta-hydroxylase gene transcription.

    PubMed

    Adachi, M; Browne, D; Lewis, E J

    2000-09-01

    The homeodomain transcription factors Arix/Phox2a and NBPhox/Phox2b play a role in the specification of the noradrenergic phenotype of central and peripheral neurons. To better understand the functions of these two factors, we have compared the genetic organization, chromosomal location, and transcriptional regulatory properties of Arix and NBPhox. The gene structure is very similar, with each gene containing three exons and two introns, extending a total of approximately 5 kb. Arix and NBPhox are unlinked in human and mouse genomes. NBPhox is located on human Chromosome 4p12 and mouse Chromosome 5, while Arix is located on human Chromosome 11q13 and mouse Chromosome 7. Both proteins bind to three sites in the promoter proximal region of the rat dopamine beta-hydroxylase gene (DBH). In vitro, Arix and NBPhox form DNA-independent multimers and exhibit cooperative binding to the DB1 regulatory element, which contains two homeodomain recognition sites. Both proteins regulate transcription from the rat DBH promoter, and transcription is synergistically increased in the presence of the protein kinase A catalytic subunit (PKA) plus either Arix or NBPhox. The transcription factors exhibit similar concentration-dependent efficacies, and when they are coexpressed, transcription is stimulated to a value approximately equal to that seen with either factor alone. The N-terminal segment of Arix is essential for transcriptional regulatory activity, and this region bears 50% identity with NBPhox, suggesting a similar mechanism of transcriptional activation of the DBH gene. We conclude from this study that Arix and NBPhox exhibit indistinguishable and independent transcriptional regulatory properties on the DBH promoter.

  9. Structural and Biophysical Insights into the Ligand-Free Pitx2 Homeodomain and a Ring Dermoid of the Cornea Inducing Homeodomain Mutant

    PubMed Central

    Doerdelmann, Thomas; Kojetin, Douglas J.; Baird-Titus, Jamie M.; Solt, Laura A.; Burris, Thomas P.; Rance, Mark

    2012-01-01

    The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. NMR techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes the autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA-binding affinity and a 5°C decrease in the thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in only a 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to other Pitx2 homeodomain mutations. PMID:22224469

  10. Dual Functions of the KNOTTED1 Homeodomain: Sequence-Specific DNA Binding and Regulation of Cell-to-Cell Transport

    USDA-ARS?s Scientific Manuscript database

    The homeodomain forms a trihelical structure, with the third helix conferring specific interactions with the DNA major groove. A specific class of plant homeodomain proteins, called KNOX [KNOTTED1 (KN1)-like homeobox], also has the ability to signal between cells by directly trafficking through inte...

  11. The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori.

    PubMed

    Ogawa, Naoki; Kishimoto, Atsuhiro; Asano, Tsunaki; Izumi, Susumu

    2005-03-01

    In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5'-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions -147 and -140. When the promoter construct mutated at positions -143, -142, and -141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.

  12. Cooperative interactions between paired domain and homeodomain.

    PubMed

    Jun, S; Desplan, C

    1996-09-01

    The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

  13. Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

    PubMed

    Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

    2015-07-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Functional analysis of a Wheat Homeodomain protein, TaR1, reveals that host chromatin remodelling influences the dynamics of the switch to necrotrophic growth in the phytopathogenic fungus Zymoseptoria tritici.

    PubMed

    Lee, Jack; Orosa, Beatriz; Millyard, Linda; Edwards, Martin; Kanyuka, Kostya; Gatehouse, Angharad; Rudd, Jason; Hammond-Kosack, Kim; Pain, Naomi; Sadanandom, Ari

    2015-04-01

    A distinguishing feature of Septoria leaf blotch disease in wheat is the long symptomless growth of the fungus amongst host cells followed by a rapid transition to necrotrophic growth resulting in disease lesions. Global reprogramming of host transcription marks this switch to necrotrophic growth. However no information exists on the components that bring about host transcriptional reprogramming. Gene-silencing, confocal-imaging and protein-protein interaction assays where employed to identify a plant homeodomain (PHD) protein, TaR1 in wheat that plays a critical role during the transition from symptomless to necrotrophic growth of Septoria. TaR1-silenced wheat show earlier symptom development upon Septoria infection but reduced fungal sporulation indicating that TaR1 is key for prolonging the symptomless phase and facilitating Septoria asexual reproduction. TaR1 is localized to the nucleus and binds to wheat Histone 3. Trimethylation of Histone 3 at lysine 4 (H3K4) and lysine 36 (H3K36) are found on open chromatin with actively transcribed genes, whereas methylation of H3K27 and H3K9 are associated with repressed loci. TaR1 specifically recognizes dimethylated and trimethylated H3K4 peptides suggesting that it regulates transcriptional activation at open chromatin. We conclude that TaR1 is an important component for the pathogen life cycle in wheat that promotes successful colonization by Septoria. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  15. Analysis of homeodomain function by structure-based design of a transcription factor.

    PubMed Central

    Pomerantz, J L; Pabo, C O; Sharp, P A

    1995-01-01

    The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits. Images Fig. 1 Fig. 2 Fig. 4 PMID:7568211

  16. A natural product from Cannabis sativa subsp. sativa inhibits homeodomain-interacting protein kinase 2 (HIPK2), attenuating MPP(+)-induced apoptosis in human neuroblastoma SH-SY5Y cells.

    PubMed

    Wang, Guan; Zhu, Lingjuan; Zhao, Yuqian; Gao, Suyu; Sun, Dejuan; Yuan, Jingquan; Huang, Yuxin; Zhang, Xue; Yao, Xinsheng

    2017-03-30

    Homeodomain-interacting protein kinase 2 (HIPK2) is a conserved serine/threonine kinase, which regulate transcription, cell differentiation, proliferation and apoptosis. Previous evidences indicated that HIPK2 could be involved in the pathogenesis of neurodegenerative diseases, suggesting as a novel target for Parkinson's disease (PD) therapeutic development. Herein, gene microarray analysis was performed to verify the key regulatory function of HIPK2 in PD. (Z)-methylp-hydroxycinnamate (ZMHC, 7) with other eighteen compounds were isolated from Cannabis sativa subsp. sativa, growing in Bama Yao Autonomous County, one of the five largest longevity regions of the world. Intriguingly, ZMHC was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics (MD) simulations. Moreover, cell morphology, flow cytometry and western blot assay suggested that ZMHC inhibited HIPK2, which attenuated MPP(+)-induced apoptosis in SH-SY5Y cells. In conclusion, these findings discovered a natural product that inhibited HIPK2, and highlighted that ZMHC could be a potential precursor agent for future PD therapy.

  17. Lock and key binding of the HOX YPWM peptide to the PBX homeodomain.

    PubMed

    Sprules, Tara; Green, Nancy; Featherstone, Mark; Gehring, Kalle

    2003-01-10

    HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.

  18. Lock and Key Binding of the HOX YPWM Peptide to the PBX Homeodomain

    SciTech Connect

    Sprules, Tara; Green, N.; Featherstone, M.; Gehring, Kalle

    2003-01-10

    HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.

  19. Functional specificity of Hoxa-4 in vertebral patterning lies outside of the homeodomain.

    PubMed Central

    Sreenath, T L; Pollock, R A; Bieberich, C J

    1996-01-01

    The Hox family of proteins plays a central role in establishing the body plan of a wide range of metazoan organisms. Each member of this family of transcriptional regulators has a distinct functional specificity, yet they bind to similar DNA target sequences through their conserved homeodomain. The mechanisms whereby Hox proteins achieve their diverse specificities in vivo remain undefined. Using the opposing effects of Hoxa-4 and Hoxc-8 in vertebral patterning, we demonstrate by replacing the homeodomain of Hoxa-4 with that of Hoxc-8 that the functional specificity of Hoxa-4 does not track with the homeodomain. These observations provide evidence that other regions of Hox proteins play an important role in mediating functional specificity during mammalian embryogenesis. Images Fig. 1 Fig. 2 PMID:8790382

  20. Disordered tails of homeodomains facilitate DNA recognition by providing a trade-off between folding and specific binding.

    PubMed

    Tóth-Petróczy, Agnes; Simon, Istvan; Fuxreiter, Monika; Levy, Yaakov

    2009-10-28

    DNA binding specificity of homeodomain transcription factors is critically affected by disordered N-terminal tails (N-tails) that undergo a disorder-to-order transition upon interacting with DNA. The mechanism of the binding process and the molecular basis of selectivity are largely unknown. The coupling between folding and DNA binding of Antp and NK-2 homeodomains was investigated by coarse-grained molecular dynamics simulations using the native protein-DNA complex. The disordered N-tails were found to decrease the stability of the free proteins by competing with the native intramolecular interactions and increasing the radius of gyration of the homeodomain cores. In the presence of DNA, however, the N-tails increase the stability of the homeodomains by reducing the coupling between folding and DNA binding. Detailed studies on Antp demonstrate that the N-tail anchors the homeodomain to DNA and accelerates formation of specific interactions all along the protein-DNA interface. The tidal electrostatic forces between the N-tail and DNA induce faster and tighter binding of the homeodomain core to the DNA; this mechanism conforms to a fly-casting mechanism. In agreement with experiments, the N-tail of Antp also improves the binding affinity for DNA, with a major contribution by the released waters. These results imply that varying the degree of folding upon binding and thereby modulating the size of the buried surface-disordered N-tails of homeodomains can fine-tune the binding strength for specific DNA sequences. Overall, both the kinetics and thermodynamics of specific DNA binding by homeodomains can be improved by N-tails using a mechanism that is inherent in their disordered state.

  1. Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos.

    PubMed Central

    Dessain, S; Gross, C T; Kuziora, M A; McGinnis, W

    1992-01-01

    Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities. Images PMID:1347746

  2. Crystal structure of the human NKX2.5 homeodomain in complex with DNA target.

    PubMed

    Pradhan, Lagnajeet; Genis, Caroli; Scone, Peyton; Weinberg, Ellen O; Kasahara, Hideko; Nam, Hyun-Joo

    2012-08-14

    NKX2.5 is a homeodomain containing transcription factor regulating cardiac formation and function, and its mutations are linked to congenital heart disease. Here we provide the first report of the crystal structure of the NKX2.5 homeodomain in complex with double-stranded DNA of its endogenous target, locating within the proximal promoter -242 site of the atrial natriuretic factor gene. The crystal structure, determined at 1.8 Å resolution, demonstrates that NKX2.5 homeodomains occupy both DNA binding sites separated by five nucleotides without physical interaction between themselves. The two homeodomains show identical conformation despite the differences in the DNA sequences they bind, and no significant bending of the DNA was observed. Tyr54, absolutely conserved in NK2 family proteins, mediates sequence-specific interaction with the TAAG motif. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.

  3. Crystal Structure of the Human NKX2.5 Homeodomain in Complex with DNA Target

    SciTech Connect

    Pradhan, Lagnajeet; Genis, Caroli; Scone, Peyton; Weinberg, Ellen O.; Kasahara, Hideko; Nam, Hyun-Joo

    2012-10-16

    NKX2.5 is a homeodomain containing transcription factor regulating cardiac formation and function, and its mutations are linked to congenital heart disease. Here we provide the first report of the crystal structure of the NKX2.5 homeodomain in complex with double-stranded DNA of its endogenous target, locating within the proximal promoter -242 site of the atrial natriuretic factor gene. The crystal structure, determined at 1.8 {angstrom} resolution, demonstrates that NKX2.5 homeodomains occupy both DNA binding sites separated by five nucleotides without physical interaction between themselves. The two homeodomains show identical conformation despite the differences in the DNA sequences they bind, and no significant bending of the DNA was observed. Tyr54, absolutely conserved in NK2 family proteins, mediates sequence-specific interaction with the TAAG motif. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.

  4. Homeodomain transcription factors regulate BMP-2-induced osteoactivin transcription in osteoblasts.

    PubMed

    Singh, Maneet; Del Carpio-Cano, Fabiola E; Monroy, M Alexandra; Popoff, Steven N; Safadi, Fayez F

    2012-01-01

    Osteoactivin (OA) is required for the differentiation of osteoblast cells. OA expression is stimulated by bone morphogenetic protein-2 (BMP-2). BMP-2 recruits homeodomain transcription factors Dlx3, Dlx5, and Msx2 to selectively activate or repress transcription of osteogenic genes and hence tightly regulate their transcription during osteoblast differentiation. Considering the key roles of Dlx3, Dlx5, and Msx2 in osteoblast differentiation, here we hypothesize that homeodomain proteins regulate BMP-2-induced OA transcription during osteoblast differentiation. Four classical homeodomain binding sites were identified in the proximal 0.96 kb region of rat OA promoter. Deletions and mutagenesis studies of the OA promoter region indicated that all four homeodomain binding sites are crucial for BMP-2-induced OA promoter activity. Simultaneous disruption of homeodomain binding sites at -852 and -843 of the transcription start site of OA gene significantly decreased the BMP-2-induced OA transcription and inhibited binding of Dlx3, Dlx5, and Msx2 proteins to the OA promoter. Dlx3 and Dlx5 proteins were found to activate the OA transcription, whereas, Msx2 suppressed BMP-2-induced OA transcription. Using chromatin immunoprecipitation assays, we demonstrated that the OA promoter is predominantly occupied by Dlx3 and Dlx5 during the proliferation and matrix maturation stages of osteoblast differentiation, respectively. During the matrix mineralization stage, BMP-2 robustly enhanced the recruitment of Dlx5 and to a lesser extent of Dlx3 and Msx2 to the OA promoter region. Collectively, our results show that the BMP-2-induced OA transcription is differentially regulated by Dlx3, Dlx5, and Msx2 during osteoblast differentiation.

  5. Recognition of Histone H3K4 Trimethylation by the Plant Homeodomain of PHF2 Modulates Histone Demethylation

    SciTech Connect

    Wen, Hong; Li, Jingzhi; Song, Tanjing; Lu, Ming; Kan, Pu-Yeh; Lee, Min Gyu; Sha, Bingdong; Shi, Xiaobing

    2010-10-28

    Distinct lysine methylation marks on histones create dynamic signatures deciphered by the 'effector' modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the 'cross-talk' of histone modifications.

  6. The homeodomain transcription factor Phox2 in the stellate ganglion of the squid Loligo pealei.

    PubMed

    Burbach, J Peter H; Hellemons, Anita J C G M; Grant, Philip; Pant, Harish C

    2015-06-26

    Homeodomain transcription factors regulate development of embryos and cellular physiology in adult systems. Paired-type homeodomain genes constitute a subclass that has been particularly implicated in establishment of neuronal identity in the mammalian nervous system. We isolated fragments of eight homeodomain genes of this subclass expressed in the stellate ganglion of the North Atlantic long finned squid Loligo pealei (lp) [Note: Loligo pealei has been officially renamed Doryteuthis pealei. For reasons of uniformity and clarity Loligo pealei (lp) is used here]. Of the most abundant ones, we cloned a full length cDNA which encoded the squid ortholog of the paired-type homeodomain proteins Phox2a/b. The homology of lpPhox2 to invertebrate and mammalian Phox2 was limited to the homeodomain. In contrast to mouse Phox2b, lpPhox2 was unable to transactivate the dopamine beta-hydroxylase (DBH) promoter in a heterologous mammalian transfection system. In vivo, lpPhox2 was expressed in the developing stellate ganglion of stage 27 squid embryos and continued to be expressed in the adult stellate neurons where expression was confined to the giant fiber lobe containing the neurons that form the giant axons. The expression of lpPhox was similarly timed and distributed as the Fmrf gene. Furthermore, the Fmrf upstream region contained putative Phox2a/b binding sites. These results suggest a role of lpPhox2 in the developmental specification of neuronal identity and regulation of neurons of the squid giant axon. © 2015. Published by The Company of Biologists Ltd.

  7. The homeodomain transcription factor Phox2 in the stellate ganglion of the squid Loligo pealei

    PubMed Central

    Burbach, J. Peter H.; Hellemons, Anita J. C. G. M.; Grant, Philip; Pant, Harish C.

    2015-01-01

    ABSTRACT Homeodomain transcription factors regulate development of embryos and cellular physiology in adult systems. Paired-type homeodomain genes constitute a subclass that has been particularly implicated in establishment of neuronal identity in the mammalian nervous system. We isolated fragments of eight homeodomain genes of this subclass expressed in the stellate ganglion of the North Atlantic long finned squid Loligo pealei (lp) [Note: Loligo pealei has been officially renamed Doryteuthis pealei. For reasons of uniformity and clarity Loligo pealei (lp) is used here]. Of the most abundant ones, we cloned a full length cDNA which encoded the squid ortholog of the paired-type homeodomain proteins Phox2a/b. The homology of lpPhox2 to invertebrate and mammalian Phox2 was limited to the homeodomain. In contrast to mouse Phox2b, lpPhox2 was unable to transactivate the dopamine beta-hydroxylase (DBH) promoter in a heterologous mammalian transfection system. In vivo, lpPhox2 was expressed in the developing stellate ganglion of stage 27 squid embryos and continued to be expressed in the adult stellate neurons where expression was confined to the giant fiber lobe containing the neurons that form the giant axons. The expression of lpPhox was similarly timed and distributed as the Fmrf gene. Furthermore, the Fmrf upstream region contained putative Phox2a/b binding sites. These results suggest a role of lpPhox2 in the developmental specification of neuronal identity and regulation of neurons of the squid giant axon. PMID:26116657

  8. A phosphorylation site in the ftz homeodomain is required for activity.

    PubMed Central

    Dong, J; Hung, L H; Strome, R; Krause, H M

    1998-01-01

    The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. Here we show that one of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala resulted in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein. PMID:9545243

  9. Role of conserved salt bridges in homeodomain stability and DNA binding.

    PubMed

    Torrado, Mario; Revuelta, Julia; Gonzalez, Carlos; Corzana, Francisco; Bastida, Agatha; Asensio, Juan Luis

    2009-08-28

    The sequence information available for homeodomains reveals that salt bridges connecting pairs 19/30, 31/42, and 17/52 are frequent, whereas aliphatic residues at these sites are rare and mainly restricted to proteins from homeotherms. We have analyzed the influence of salt and hydrophobic bridges at these sites on the stability and DNA binding properties of human Hesx-1 homeodomain. Regarding the protein stability, our analysis shows that hydrophobic side chains are clearly preferred at positions 19/30 and 31/42. This stabilizing influence results from the more favorable packing of the aliphatic side chains with the protein core, as illustrated by the three-dimensional solution structure of a thermostable variant, herein reported. In contrast only polar side chains seem to be tolerated at positions 17/52. Interestingly, despite the significant influence of pairs 19/30 and 31/42 on the stability of the homeodomain, their effect on DNA binding ranges from modest to negligible. The observed lack of correlation between binding strength and conformational stability in the analyzed variants suggests that salt/hydrophobic bridges at these specific positions might have been employed by evolution to independently modulate both properties.

  10. Concerted dynamics link allosteric sites in the PBX homeodomain.

    PubMed

    Farber, Patrick J; Mittermaier, Anthony

    2011-01-21

    The PBX1 homeodomain (PBX-HD) cooperatively binds DNA with Hox transcription factors and helps to regulate gene expression during vertebrate development. Allostery plays an important role in these interactions. DNA binding on one surface of PBX-HD enhances interactions with Hox proteins at a different interface. In addition, DNA binding causes a 15-residue extension at the C-terminus of PBX-HD to undergo a disorder-to-helix transition, although this region does not directly contact the DNA. Deletion of the C-terminal extension reduces both the DNA affinity of PBX-HD and the cooperativity of forming the DNA/Hox/PBX-HD ternary complex. To better understand the mechanism underlying these allosteric interactions, we used NMR relaxation dispersion dynamics experiments to characterize millisecond-timescale motions in PBX-HD over a range of temperatures. The data show that the C-terminal extension folds to form a fourth α-helix to a level of 5-10%, even in the absence of binding partners. This suggests that PBX-HD transiently preorganizes prior to binding DNA, reminiscent of the "conformational selection" model of molecular recognition. Folding of the C-terminal extension in the unbound protein is accompanied by structural rearrangements in both the DNA binding site and the Hox binding site, suggesting a possible role for these dynamics in the allosteric mechanism of PBX-HD. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Homology modeling using simulated annealing of restrained molecular dynamics and conformational search calculations with CONGEN: application in predicting the three-dimensional structure of murine homeodomain Msx-1.

    PubMed Central

    Li, H.; Tejero, R.; Monleon, D.; Bassolino-Klimas, D.; Abate-Shen, C.; Bruccoleri, R. E.; Montelione, G. T.

    1997-01-01

    We have developed an automatic approach for homology modeling using restrained molecular dynamics and simulated annealing procedures, together with conformational search algorithms available in the molecular mechanics program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168). The accuracy of the method is validated by "predicting" structures of two homeodomain proteins with known three-dimensional structures, and then applied to predict the three-dimensional structure of the homeodomain of the murine Msx-1 transcription factor. Regions of the unknown protein structure that are highly homologous to the known template structure are constrained by "homology distance constraints," whereas the conformations of nonhomologous regions of the unknown protein are defined only by the potential energy function. A full energy function (excluding explicit solvent) is employed to ensure that the calculated structures have good conformational energies and are physically reasonable. As in NMR structure determinations, information on the consistency of the structure prediction is obtained by superposition of the resulting family of protein structures. In this paper, our homology modeling algorithm is described and compared with related homology modeling methods using spatial constraints derived from the structures of homologous proteins. The software is then used to predict the DNA-bound structures of three homeodomain proteins from the X-ray crystal structure of the engrailed homeodomain protein (Kissinger CR et al., 1990, Cell 63:579-590). The resulting backbone and side-chain conformations of the modeled yeast Mat alpha 2 and D. melanogaster Antennapedia homeodomains are excellent matches to the corresponding published X-ray crystal (Wolberger C et al., 1991, Cell 67:517-528) and NMR (Billeter M et al., 1993, J Mol Biol 234:1084-1097) structures, respectively. Examination of these structures of Msx-1 reveals a network of highly conserved surface salt bridges that

  12. Proteasomal selection of multiprotein complexes recruited by LIM homeodomain transcription factors

    PubMed Central

    Güngör, Cenap; Taniguchi-Ishigaki, Naoko; Ma, Hong; Drung, Alexander; Tursun, Baris; Ostendorff, Heather P.; Bossenz, Michael; Becker, Catherina G.; Becker, Thomas; Bach, Ingolf

    2007-01-01

    Complexes composed of multiple proteins regulate most cellular functions. However, our knowledge about the molecular mechanisms governing the assembly and dynamics of these complexes in cells remains limited. The in vivo activity of LIM homeodomain (LIM-HD) proteins, a class of transcription factors that regulates neuronal development, depends on the high-affinity association of their LIM domains with cofactor of LIM homeodomain proteins (LIM-HDs) (CLIM, also known as Ldb or NLI). CLIM cofactors recruit single-stranded DNA-binding protein 1 (SSDP1, also known as SSBP3), and this interaction is important for the activation of the LIM-HD/CLIM protein complex in vivo. Here, we identify a cascade of specific protein interactions that protect LIM-HD multiprotein complexes from proteasomal degradation. In this cascade, CLIM stabilizes LIM-HDs, and SSDP1 stabilizes CLIM. Furthermore, we show that stabilizing cofactors prevent binding of ubiquitin ligases to multiple protein interaction domains in LIM-HD recruited protein complexes. Together, our results indicate a combinatorial code that selects specific multiprotein complexes via proteasomal degradation in cells with broad implications for the assembly and specificity of multiprotein complexes. PMID:17848518

  13. Roles of the LHX3 and LHX4 LIM-Homeodomain Factors in Pituitary Development

    PubMed Central

    Mullen, Rachel D.; Colvin, Stephanie C.; Hunter, Chad S.; Savage, Jesse J.; Walvoord, Emily C.; Bhangoo, Amrit P.S.; Ten, Svetlana; Weigel, Johannes; Pfäffle, Roland W.; Rhodes, Simon J.

    2007-01-01

    The LHX3 and LHX4 LIM-homeodomain transcription factors play essential roles in pituitary gland and nervous system development. Mutations in the genes encoding these regulatory proteins are associated with combined hormone deficiency diseases in humans and animal models. Patients with these diseases have complex syndromes involving short stature, and reproductive and metabolic disorders. Analyses of the features of these diseases and the biochemical properties of the LHX3 and LHX4 proteins will facilitate a better understanding of the molecular pathways that regulate the development of the specialized hormone-secreting cells of the mammalian anterior pituitary gland. PMID:17210222

  14. Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes.

    PubMed

    Gutmanas, Aleksandras; Billeter, Martin

    2004-12-01

    Four molecular dynamics simulation trajectories of complexes between the wild-type or a mutant Antennapedia homeodomain and 2 DNA sequences were generated in order to probe the mechanisms governing the specificity of DNA recognition. The starting point was published affinity measurements showing that a single protein mutation combined with a replacement of 2 base pairs yields a new high-affinity complex, whereas the other combinations, with changes on only 1 macromolecule, exhibited lower affinity. The simulations of the 4 complexes yielded fluctuating networks of interaction. On average, these networks differ significantly, explaining the switch of affinity caused by the alterations in the macromolecules. The network of mostly hydrogen-bonding interactions involving several water molecules, which was suggested both by X-ray and NMR structures of the wild-type homeodomain and its DNA operator sequence, could be reproduced in the trajectory. More interestingly, the high-affinity complex with alterations in both the protein and the DNA yielded again a dynamic but very tight network of intermolecular interactions, however, attributing a significantly stronger role to direct hydrophobic interactions at the expense of water bridges. The other 2 homeodomain-DNA complexes, with only 1 molecule altered, show on average over the trajectories a clearly reduced number of protein-DNA interactions. The observations from these simulations suggest specific experiments and thus close the circle formed by biochemical, structural, and computational studies. The shift from a water-dominated to a more "dry" interface may prove important in the design of proteins binding DNA in a specific manner.

  15. The LIM Homeodomain Transcription Factor LHX6

    PubMed Central

    Zhang, Zichao; Gutierrez, Diana; Li, Xiao; Bidlack, Felicitas; Cao, Huojun; Wang, Jianbo; Andrade, Kelsey; Margolis, Henry C.; Amendt, Brad A.

    2013-01-01

    LHX6 is a LIM-homeobox transcription factor expressed during embryogenesis; however, the molecular mechanisms regulating LHX6 transcriptional activities are unknown. LHX6 and the PITX2 homeodomain transcription factor have overlapping expression patterns during tooth and craniofacial development, and in this report, we demonstrate new transcriptional mechanisms for these factors. PITX2 and LHX6 are co-expressed in the oral and dental epithelium and epithelial cell lines. Lhx6 expression is increased in Pitx2c transgenic mice and decreased in Pitx2 null mice. PITX2 activates endogenous Lhx6 expression and the Lhx6 promoter, whereas LHX6 represses its promoter activity. Chromatin immunoprecipitation experiments reveal endogenous PITX2 binding to the Lhx6 promoter. LHX6 directly interacts with PITX2 to inhibit PITX2 transcriptional activities and activation of multiple promoters. Bimolecular fluorescence complementation assays reveal an LHX6·PITX2 nuclear interaction in living cells. LHX6 has a dominant repressive effect on the PITX2 synergistic activation with LEF-1 and β-catenin co-factors. Thus, LHX6 acts as a transcriptional repressor and represses the expression of several genes involved in odontogenesis. We have identified specific defects in incisor, molar, mandible, bone, and root development and late stage enamel formation in Lhx6 null mice. Amelogenin and ameloblastin expression is reduced and/or delayed in the Lhx6 null mice, potentially resulting from defects in dentin deposition and ameloblast differentiation. Our results demonstrate that LHX6 regulates cell proliferation in the cervical loop and promotes cell differentiation in the anterior region of the incisor. We demonstrate new molecular mechanisms for LHX6 and an interaction with PITX2 for normal craniofacial and tooth development. PMID:23229549

  16. Repression of Virus-Induced Interferon A Promoters by Homeodomain Transcription Factor Ptx1

    PubMed Central

    Lopez, Sébastien; Island, Marie-Laure; Drouin, Jacques; Bandu, Marie-Thérese; Christeff, Nicolas; Darracq, Nicole; Barbey, Régine; Doly, Janine; Thomas, Dominique; Navarro, Sébastien

    2000-01-01

    Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE). The functional feature of the DNRE is to specifically act by repression of VRE-A activity. With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1). Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE. Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression. After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes. These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor. PMID:11003649

  17. Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19

    PubMed Central

    Boube, Muriel; Hudry, Bruno; Immarigeon, Clément; Carrier, Yannick; Bernat-Fabre, Sandra; Merabet, Samir; Graba, Yacine; Bourbon, Henri-Marc; Cribbs, David L.

    2014-01-01

    Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded “co-factor” that acts together with Hox proteins through their homeodomains in regulated developmental transcription. PMID:24786462

  18. Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding

    PubMed Central

    Proudfoot, Andrew; Geralt, Michael; Elsliger, Marc-Andre; Wilson, Ian A.; Wüthrich, Kurt; Serrano, Pedro

    2016-01-01

    SUMMARY The Gastrulation Brain Homeobox 1 (Gbx1) gene encodes the Gbx1 homeodomain that targets TAATTA motifs in dsDNA. Residues Glu17 and Arg52 in Gbx1 form a salt bridge, which is preserved in crystal structures and MD simulations of homologous homeodomain–DNA complexes. In contrast, our NMR studies show that DNA-binding to Gbx1 induces dynamic local polymorphisms, which include breaking of the Glu17–Arg52 salt bridge. To study this interaction, we produced a variant with Glu17Arg and Arg52Glu mutations, which exhibited the same fold as the wild-type protein, but a two-fold reduction in affinity for dsDNA. Analysis of the NMR structures of the Gbx1 homeodomain in the free form, the Gbx1[E17R,R52E] variant, and a Gbx1 homeodomain–DNA complex showed that stabilizing interactions of the Arg52 side chain with the DNA backbone are facilitated by transient breakage of the Glu17–Arg52 salt bridge in the DNA-bound Gbx1. PMID:27396829

  19. Comprehensive analysis of the homeodomain-leucine zipper IV transcription factor family in Cucumis sativus.

    PubMed

    Fu, Rao; Liu, Wei; Li, Qiang; Li, Jing; Wang, Lina; Ren, Zhonghai

    2013-07-01

    The class IV homeodomain-leucine zipper (HD-Zip IV) proteins are plant-specific transcriptional factors known to play crucial roles in plant growth and development. In this study, 11 cucumber (Cucumis sativus) HD-Zip IV genes were identified in the version 2 cucumber genome and found to be distributed unevenly across the chromosomes. The CsHDZIV (Cucumis sativus homeodomain-leucine zipper IV) gene family is smaller than in other studied species (except for rice) because of the absence of gene duplication events. Phylogenetic analysis showed that HD-Zip IV genes from cucumber, Arabidopsis, tomato, cotton, maize, and rice could be classified into five subgroups. All CsHDZIV genes appear to be derived from a basic module containing 11 exons in the coding region. Two conserved motifs of 21 and 19 nucleotides were found in the 3'-untranslated regions of six CsHDZIV genes, suggesting that post-transcriptional regulation may play a role in regulation of CsHDZIV genes. In addition, 6 of 11 CsHDZIV genes were found to undergo alternative splicing events. Reverse transcription PCR analysis showed that all CsHDZIV genes (except one) were expressed and showed preferential expression in reproductive organs.

  20. Discovery, optimization and validation of an optimal DNA-binding sequence for the Six1 homeodomain transcription factor.

    PubMed

    Liu, Yubing; Nandi, Soumyadeep; Martel, André; Antoun, Alen; Ioshikhes, Ilya; Blais, Alexandre

    2012-09-01

    The Six1 transcription factor is a homeodomain protein involved in controlling gene expression during embryonic development. Six1 establishes gene expression profiles that enable skeletal myogenesis and nephrogenesis, among others. While several homeodomain factors have been extensively characterized with regards to their DNA-binding properties, relatively little is known of the properties of Six1. We have used the genomic binding profile of Six1 during the myogenic differentiation of myoblasts to obtain a better understanding of its preferences for recognizing certain DNA sequences. DNA sequence analyses on our genomic binding dataset, combined with biochemical characterization using binding assays, reveal that Six1 has a much broader DNA-binding sequence spectrum than had been previously determined. Moreover, using a position weight matrix optimization algorithm, we generated a highly sensitive and specific matrix that can be used to predict novel Six1-binding sites with highest accuracy. Furthermore, our results support the idea of a mode of DNA recognition by this factor where Six1 itself is sufficient for sequence discrimination, and where Six1 domains outside of its homeodomain contribute to binding site selection. Together, our results provide new light on the properties of this important transcription factor, and will enable more accurate modeling of Six1 function in bioinformatic studies.

  1. Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain.

    PubMed

    Ju, Jeong Ho; Maeng, Jin-Soo; Lee, Duck-Yeon; Piszczek, Grzegorz; Gelmann, Edward P; Gruschus, James M

    2009-11-10

    NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.

  2. The eyeless homeodomain is dispensable for eye development in Drosophila

    PubMed Central

    Punzo, Claudio; Kurata, Shoichiro; Gehring, Walter J.

    2001-01-01

    Pax-6 genes, known to be essential for eye development, encode an evolutionarily conserved transcription factor with two DNA-binding domains. To corroborate the contribution of each DNA-binding domain to eye formation, we generated truncated forms of the Drosophila Pax-6 gene eyeless and tested their capacity to rescue the ey2 mutant. Surprisingly, EY deleted of the homeodomain rescued the ey2 mutant and triggered ectopic eyes morphogenesis. In contrast, EY lacking the paired domain failed to rescue the ey2 mutant, led to truncation of appendages, and repressed Distal-less when misexpressed. This result suggests distinct functions mediated differentially by the two DNA-binding domains of eyeless. PMID:11445545

  3. Predicting the Folding Pathway of Engrailed Homeodomain with a Probabilistic Roadmap Enhanced Reaction-Path Algorithm☆

    PubMed Central

    Li, Da-wei; Yang, Haijun; Han, Li; Huo, Shuanghong

    2008-01-01

    To predict a protein-folding pathway, we present an alternative to the time-consuming dynamic simulation of atomistic models. We replace the actual dynamic simulation with variational optimization of a reaction path connecting known initial and final protein conformations in such a way as to maximize an estimate of the reactive flux or minimize the mean first passage time at a given temperature, referred to as MaxFlux. We solve the MaxFlux global optimization problem with an efficient graph-theoretic approach, the probabilistic roadmap method (PRM). We employed CHARMM19 and the EEF1 implicit solvation model to describe the protein solution. The effectiveness of our MaxFlux-PRM is demonstrated in our promising simulation results on the folding pathway of the engrailed homeodomain. Our MaxFlux-PRM approach provides the direct evidence to support that the previously reported intermediate state is a genuine on-pathway intermediate, and the demand of CPU power is moderate. PMID:18024496

  4. Dlx5 Homeodomain: DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

    SciTech Connect

    Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; Fletterick, Robert J.; Yumoto, Fumiaki; Deacon, Ashley M.; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt; Serrano, Pedro

    2016-01-29

    The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked to SHFM-1; this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.

  5. Plant transcription factors from the homeodomain-leucine zipper family I. Role in development and stress responses.

    PubMed

    Perotti, María Florencia; Ribone, Pamela Anahí; Chan, Raquel Lía

    2017-05-01

    In front of stressful conditions plants display adaptation mechanisms leading to changes in their morphology, physiology, development and molecular composition. Transcription factors (TFs) play crucial roles in these complex adaptation processes. This work is focused in the homeodomain-leucine zipper I (HD-Zip I) family of TFs, unique to plants. First discovered in 1991, they were identified and isolated from monocotyledonous and dicotyledonous plants showing high structural similarity and diversified functions. These TFs have, besides the homeodomain and leucine zipper, conserved motifs in their carboxy-termini allowing the interaction with the basal machinery and with other regulatory proteins. The model dicotyledonous plant Arabidopsis thaliana has 17 HD-Zip I members; most of them regulated by external stimuli and hormones. These TFs are involved in key developmental processes like root and stem elongation, rosette leaves morphology determination, inflorescence stem branching, flowering and pollen hydration. Moreover, they are key players in responses to environmental stresses and illumination conditions. Several HD-Zip I encoding genes from different species were protected in patents because their overexpression or mutation generates improved agronomical phenotypes. Here we discuss many aspects about these TFs including structural features, biological functions and their utilization as biotechnological tools to improve crops. © 2017 IUBMB Life, 69(5):280-289, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  6. Dlx5 Homeodomain: DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

    DOE PAGES

    Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; ...

    2016-01-29

    The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked to SHFM-1;more » this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.« less

  7. A molecular code dictates sequence-specific DNA recognition by homeodomains.

    PubMed Central

    Damante, G; Pellizzari, L; Esposito, G; Fogolari, F; Viglino, P; Fabbro, D; Tell, G; Formisano, S; Di Lauro, R

    1996-01-01

    Most homeodomains bind to DNA sequences containing the motif 5'-TAAT-3'. The homeodomain of thyroid transcription factor 1 (TTF-1HD) binds to sequences containing a 5'-CAAG-3' core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF-1HD by both structural and functional approaches. As already suggested by the three-dimensional structure of TTF-1HD, the DNA binding specificities of the TTF-1, Antennapedia and Engrailed homeodomains, either wild-type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3' end of the core motif 5'-NAAN-3'. The nucleotide at the 5' position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF-1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule. Images PMID:8890172

  8. Sequence-specific DNA recognition by the thyroid transcription factor-1 homeodomain.

    PubMed Central

    Damante, G; Fabbro, D; Pellizzari, L; Civitareale, D; Guazzi, S; Polycarpou-Schwartz, M; Cauci, S; Quadrifoglio, F; Formisano, S; Di Lauro, R

    1994-01-01

    The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1HD within the protein-DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gln at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts. Images PMID:7915030

  9. LIM-homeodomain transcription factor Isl-1 mediates kisspeptin's effect on insulin secretion in mice.

    PubMed

    Chen, Juan; Fu, Rui; Cui, Yan; Pan, Jirong; Li, Yushan; Zhang, Xiaoxin; Evans, Sylvia M; Cui, Sheng; Liu, Jiali

    2014-08-01

    Kisspeptin and the G protein-coupled receptor 54 (GPR54) are highly abundant in the pancreas. In addition, circulating kisspeptin directly influences insulin secretion through GPR54. However, the mechanisms by which kisspeptin affects insulin release are unclear. The LIM-homeodomain transcription factor, Isl-1, is expressed in all pancreatic islet cells and is involved in regulating both islet development and insulin secretion. We therefore investigated potential interactions between kisspeptin and Isl-1. Our results demonstrate that Isl-1 and GPR54 are coexpressed in mouse pancreatic islet β-cells and NIT cells. Both in vitro and in vivo results demonstrate that kisspeptin-54 (KISS-54) inhibits Isl-1 expression and insulin secretion and both the in vivo and in vitro effects of KISS-54 on insulin gene expression and secretion are abolished when an Isl-1-inducible knockout model is used. Moreover, our results demonstrate that the direct action of KISS-54 on insulin secretion is mediated by Isl-1. Our results further show that KISS-54 influences Isl-1 expression and insulin secretion through the protein kinase C-ERK1/2 pathway. Conversely, insulin has a feedback loop via the Janus kinase-phosphatidylinositol 3-kinase pathway regulating kisspeptin expression and secretion. These findings are important in understanding mechanisms of insulin secretion and metabolism in diabetes.

  10. A conserved C-terminal domain in PBX increases DNA binding by the PBX homeodomain and is not a primary site of contact for the YPWM motif of HOXA1.

    PubMed

    Green, N C; Rambaldi, I; Teakles, J; Featherstone, M S

    1998-05-22

    HOX proteins are dependent upon cofactors of the PBX family for specificity of DNA binding. Two regions that have been implicated in HOX/PBX cooperative interactions are the YPWM motif, found N-terminal to the HOX homeodomain, and the GKFQ domain (also known as the Hox cooperativity motif) immediately C-terminal to the PBX homeodomain. Using derivatives of the E2A-PBX oncoprotein, we find that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to the stability of the complex. By contrast, the YPWM motif is strictly required for cooperative interactions in vitro and in vivo, even with mutants of E2A-PBX lacking the GKFQ domain. Using truncated PBX proteins, we show that the YPWM motif contacts the PBX homeodomain. The presence of the GKFQ domain increases monomer binding by the PBX homeodomain 5-fold, and the stability of the HOXA1.E2A-PBX complex 2-fold. These data suggest that the GKFQ domain acts mainly to increase DNA binding by PBX, rather than providing a primary contact site for the YPWM motif of HOXA1. We have identified 2 residues, Glu-301 and Tyr-305, required for GKFQ function and suggest that this is dependent on alpha-helical character.

  11. Local folding and misfolding in the PBX homeodomain from a three-state analysis of CPMG relaxation dispersion NMR data.

    PubMed

    Farber, Patrick J; Slager, Jelle; Mittermaier, Anthony K

    2012-08-30

    NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments represent a powerful approach for characterizing protein internal motions and for gaining insight into fundamental biological processes such as protein folding, catalysis, and allostery. In most cases, CPMG data are analyzed assuming that the protein exchanges between two different conformational states. Systems exchanging among more than two states are far more challenging to characterize by CPMG NMR. For example, in the case of three-state exchange in the fast time scale regime, it is difficult to uniquely connect the parameters extracted from CPMG analyses with the physical parameters of most interest, intercoversion rates, populations, and chemical shift differences for exchanging states. We have developed a grid search selection procedure that allows these physical parameters to be uniquely determined from CPMG data, based on additional information, which in this study comprises ligand-induced chemical shift perturbations. We applied this approach to the PBX homeodomain (PBX-HD), a three-helix protein with a C-terminal extension that folds into a fourth helix upon binding to DNA. We recently showed that the C-terminal extension transiently folds, even in the absence DNA, in a process that is likely tied to the cooperative binding of PBX-HD to DNA and other homeodomains. Using the grid search selection procedure, we found that PBX-HD undergoes exchange between three different conformational states, a major form in which the C-terminal extension is unfolded, the previously identified state in which the C-terminal extension forms a fourth helix, and an additional state in which the C-terminal extension is misfolded.

  12. Exploring the DNA-recognition potential of homeodomains.

    PubMed

    Chu, Stephanie W; Noyes, Marcus B; Christensen, Ryan G; Pierce, Brian G; Zhu, Lihua J; Weng, Zhiping; Stormo, Gary D; Wolfe, Scot A

    2012-10-01

    The recognition potential of most families of DNA-binding domains (DBDs) remains relatively unexplored. Homeodomains (HDs), like many other families of DBDs, display limited diversity in their preferred recognition sequences. To explore the recognition potential of HDs, we utilized a bacterial selection system to isolate HD variants, from a randomized library, that are compatible with each of the 64 possible 3' triplet sites (i.e., TAANNN). The majority of these selections yielded sets of HDs with overrepresented residues at specific recognition positions, implying the selection of specific binders. The DNA-binding specificity of 151 representative HD variants was subsequently characterized, identifying HDs that preferentially recognize 44 of these target sites. Many of these variants contain novel combinations of specificity determinants that are uncommon or absent in extant HDs. These novel determinants, when grafted into different HD backbones, produce a corresponding alteration in specificity. This information was used to create more explicit HD recognition models, which can inform the prediction of transcriptional regulatory networks for extant HDs or the engineering of HDs with novel DNA-recognition potential. The diversity of recovered HD recognition sequences raises important questions about the fitness barrier that restricts the evolution of alternate recognition modalities in natural systems.

  13. Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes

    SciTech Connect

    Shi, Ge; Sohn, Kyung-Cheol; Choi, Tae-Young; Choi, Dae-Kyoung; Lee, Sang-Sin; Ou, Bai-sheng; Kim, Sooil; Lee, Young Ho; Yoon, Tae-Jin; Kim, Seong-Jin; Lee, Young; Seo, Young-Joon; Lee, Jeung-Hoon; Kim, Chang Deok

    2010-11-15

    Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

  14. Genetic Characterization of the Homeodomain-Independent Activity of the Drosophila Fushi Tarazu Gene Product

    PubMed Central

    Hyduk, D.; Percival-Smith, A.

    1996-01-01

    The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results. PMID:8852847

  15. Mapping of the ARIX homeodomain gene to mouse chromosome 7 and human chromosome 11q13

    SciTech Connect

    Johnson, K.R.; Smith, L.; Rhodes, J.

    1996-05-01

    The recently described homeodomain protein ARIX is expressed specifically in noradreneric cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine {beta}-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouse Arix was positioned approximately 50 cM distal to the centromere of chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 11q13.3-q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.

  16. Alternative Splicing of the LIM-Homeodomain Transcription Factor Isl1 in the Mouse Retina

    PubMed Central

    Whitney, Irene E.; Kautzman, Amanda G.; Reese, Benjamin E.

    2015-01-01

    Islet-1 (Isl1) is a LIM-homeodomain (LIM-HD) transcription factor that functions in a combinatorial manner with other LIM-HD proteins to direct the differentiation of distinct cell types within the central nervous system and many other tissues. A study of pancreatic cell lines showed that Isl1 is alternatively spliced generating a second isoform, Isl1β, which is missing 23 amino acids within the C-terminal region. This study examines the expression of the canonical and alternative Isl1 transcripts across other tissues, in particular, within the retina, where Isl1 is required for the differentiation of multiple neuronal cell types. The alternative splicing of Isl1 is shown to occur in multiple tissues, but the relative abundance of Isl1α and Isl1 β expression varies greatly across them. In most tissues, Isl1α is the more abundant transcript, but in others the transcripts are expressed equally, or the alternative splice variant is dominant. Within the retina, differential expression of the two Isl1 transcripts increases as a function of development, with dynamic changes in expression peaking at E16.5 and again at P10. At the cellular level, individual retinal ganglion cells vary in their expression, with a subset of small-to-medium sized cells expressing only the alternative isoform. The functional significance of the difference in protein sequence between the two Isl1 isoforms was also assessed using a luciferase assay, demonstrating that the alternative isoform forms a less effective transcriptional complex for activating gene expression. These results demonstrate the differential presence of the canonical and alternative isoforms of Isl1 amongst retinal ganglion cell classes. As Isl1 participates in the differentiation of multiple cell types within the CNS, the present results support a role for alternative splicing in the establishment of cellular diversity in the developing nervous system. PMID:25752730

  17. Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

    SciTech Connect

    Bhati, Mugdha; Lee, Christopher; Nancarrow, Amy L.; Lee, Mihwa; Craig, Vanessa J.; Bach, Ingolf; Guss, J. Mitchell; Mackay, Joel P.; Matthews, Jacqueline M.

    2008-09-03

    LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1{sub LBD}). Although the LIM interaction domain of Ldb1 (Ldb1{sub LID}) and Isl1{sub LBD} share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1{sub LBD} mimics Ldb1{sub LID}. These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

  18. The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

    PubMed Central

    Ohlenschläger, Oliver; Kuhnert, Anja; Schneider, Annerose; Haumann, Sebastian; Bellstedt, Peter; Keller, Heidi; Saluz, Hans-Peter; Hortschansky, Peter; Hänel, Frank; Grosse, Frank; Görlach, Matthias; Pospiech, Helmut

    2012-01-01

    The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund–Thomson, RAPADILINO and Baller–Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth. We have identified the first 54 amino acids of RecQL4 (RecQL4_N54) as the minimum interaction region with human TopBP1. The solution structure of RecQL4_N54 was determined by heteronuclear liquid–state nuclear magnetic resonance (NMR) spectroscopy (PDB 2KMU; backbone root-mean-square deviation 0.73 Å). Despite low-sequence homology, the well-defined structure carries an overall helical fold similar to homeodomain DNA-binding proteins but lacks their archetypical, minor groove-binding N-terminal extension. Sequence comparison indicates that this N-terminal homeodomain-like fold is a common hallmark of metazoan RecQL4 and yeast Sld2 DNA replication initiation factors. RecQL4_N54 binds DNA without noticeable sequence specificity yet with apparent preference for branched over double-stranded (ds) or single-stranded (ss) DNA. NMR chemical shift perturbation observed upon titration with Y-shaped, ssDNA and dsDNA shows a major contribution of helix α3 to DNA binding, and additional arginine side chain interactions for the ss and Y-shaped DNA. PMID:22730300

  19. The expression pattern of the murine Hoxa-10 gene and the sequence recognition of its homeodomain reveal specific properties of Abdominal B-like genes.

    PubMed Central

    Benson, G V; Nguyen, T H; Maas, R L

    1995-01-01

    Homeobox genes of the Abdominal B (AbdB) family constitute a distinct subset of vertebrate Hox genes. Analysis of the murine Hoxa-10 gene, one member of this family, revealed several properties specific to this class. Two transcripts of Hoxa-10, a10-1 and a10-2, encode homeodomain proteins of 55 kDa (399 amino acids) and 16 kDa (96 amino acids), respectively. These proteins have identical homeodomains and C-terminal regions encoded by a common 3' exon but differ significantly in the sizes of their N-terminal regions because of the usage of alternative 5' exons. The 5' exon of the a10-2 form is also present in transcripts of Hoxa-9, the next 3' gene, indicating that splicing can occur between adjacent AbdB Hox genes within a cluster. Both Hoxa-10 transcripts demonstrated identical patterns of expression in the posterior body and proximal limb bud, differentiating them from AbdB morphogenetic and regulatory transcripts and suggesting a role with other AbdB Hox genes in the patterning of these structures. Finally, a binding site selection identified the sequence AA(A/T)TTTTATTAC as the Hoxa-10 homeodomain consensus binding site, with a TTAT core sequence. Preferential recognition of a TTAT core therefore differentiates the AbdB class from Antennapedia (Antp) class gene products which bind a TAAT core. Thus, in vertebrates, structural similarities, coordinate transcriptional regulation, sites of expression, and binding site preferences all serve to distinguish AbdB from Antp Hox genes. PMID:7862151

  20. Structural insight into coordinated recognition of trimethylated histone H3 lysine 9 (H3K9me3) by the plant homeodomain (PHD) and tandem tudor domain (TTD) of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) protein.

    PubMed

    Cheng, Jingdong; Yang, Yi; Fang, Jian; Xiao, Jianxiong; Zhu, Tingting; Chen, Fei; Wang, Ping; Li, Ze; Yang, Huirong; Xu, Yanhui

    2013-01-11

    UHRF1 is an important epigenetic regulator connecting DNA methylation and histone methylations. UHRF1 is required for maintenance of DNA methylation through recruiting DNMT1 to DNA replication forks. Recent studies have shown that the plant homeodomain (PHD) of UHRF1 recognizes the N terminus of unmodified histone H3, and the interaction is inhibited by methylation of H3R2, whereas the tandem tudor domain (TTD) of UHRF1 recognizes trimethylated histone H3 lysine 9 (H3K9me3). However, how the two domains of UHRF1 coordinately recognize histone methylations remains elusive. In this report, we identified that PHD largely enhances the interaction between TTD and H3K9me3. We present the crystal structure of UHRF1 containing both TTD and PHD (TTD-PHD) in complex with H3K9m3 peptide at 3.0 Å resolution. The structure shows that TTD-PHD binds to the H3K9me3 peptide with 1:1 stoichiometry with the two domains connected by the H3K9me3 peptide and a linker region. The TTD interacts with residues Arg-8 and trimethylated Lys-9, and the PHD interacts with residues Ala-1, Arg-2, and Lys-4 of the H3K9me3 peptide. The biochemical experiments indicate that PHD-mediated recognition of unmodified H3 is independent of the TTD, whereas TTD-mediated recognition of H3K9me3 PHD. Thus, both TTD and PHD are essential for specific recognition of H3K9me3 by UHRF1. Interestingly, the H3K9me3 peptide induces conformational changes of TTD-PHD, which do not affect the autoubiquitination activity or hemimethylated DNA binding affinity of UHRF1 in vitro. Taken together, our studies provide structural insight into the coordinated recognition of H3K9me3 by the TTD and PHD of UHRF1.

  1. Crystallization and preliminary X-ray analysis of the NKX2.5 homeodomain in complex with DNA.

    PubMed

    Genis, Caroli; Scone, Peyton; Kasahara, Hideko; Nam, Hyun Joo

    2008-11-01

    As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the -242 promoter region of atrial natriuretic factor. Crystals of the homeodomain-DNA complex diffracted X-rays to 1.7 A resolution and belonged to space group P6(5), with unit-cell parameters a = b = 71.5, c = 94.3 A. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.

  2. The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation.

    PubMed

    Mitton, K P; Swain, P K; Chen, S; Xu, S; Zack, D J; Swaroop, A

    2000-09-22

    Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function.

  3. Genome-wide analysis of the homeodomain-leucine zipper (HD-ZIP) gene family in peach (Prunus persica).

    PubMed

    Zhang, C H; Ma, R J; Shen, Z J; Sun, X; Korir, N K; Yu, M L

    2014-04-08

    In this study, 33 homeodomain-leucine zipper (HD-ZIP) genes were identified in peach using the HD-ZIP amino acid sequences of Arabidopsis thaliana as a probe. Based on the phylogenetic analysis and the individual gene or protein characteristics, the HD-ZIP gene family in peach can be classified into 4 subfamilies, HD-ZIP I, II, III, and IV, containing 14, 7, 4, and 8 members, respectively. The most closely related peach HD-ZIP members within the same subfamilies shared very similar gene structure in terms of either intron/exon numbers or lengths. Almost all members of the same subfamily shared common motif compositions, thereby implying that the HD-ZIP proteins within the same subfamily may have functional similarity. The 33 peach HD-ZIP genes were distributed across scaffolds 1 to 7. Although the primary structure varied among HD-ZIP family proteins, their tertiary structures were similar. The results from this study will be useful in selecting candidate genes from specific subfamilies for functional analysis.

  4. Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif.

    PubMed

    Voelzmann, André; Wulf, Anna-Lena; Eckardt, Franka; Thielisch, Melanie; Brondolin, Mirco; Pesch, Yanina-Yasmin; Sociale, Mariangela; Bauer, Reinhard; Hoch, Michael

    2016-04-01

    Drosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-β import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.

  5. START lipid/sterol-binding domains are amplified in plants and are predominantly associated with homeodomain transcription factors.

    PubMed

    Schrick, Kathrin; Nguyen, Diana; Karlowski, Wojciech M; Mayer, Klaus F X

    2004-01-01

    In animals, steroid hormones regulate gene expression by binding to nuclear receptors. Plants lack genes for nuclear receptors, yet genetic evidence from Arabidopsis suggests developmental roles for lipids/sterols analogous to those in animals. In contrast to nuclear receptors, the lipid/sterol-binding StAR-related lipid transfer (START) protein domains are conserved, making them candidates for involvement in both animal and plant lipid/sterol signal transduction. We surveyed putative START domains from the genomes of Arabidopsis, rice, animals, protists and bacteria. START domains are more common in plants than in animals and in plants are primarily found within homeodomain (HD) transcription factors. The largest subfamily of HD-START proteins is characterized by an HD amino-terminal to a plant-specific leucine zipper with an internal loop, whereas in a smaller subfamily the HD precedes a classic leucine zipper. The START domains in plant HD-START proteins are not closely related to those of animals, implying collateral evolution to accommodate organism-specific lipids/sterols. Using crystal structures of mammalian START proteins, we show structural conservation of the mammalian phosphatidylcholine transfer protein (PCTP) START domain in plants, consistent with a common role in lipid transport and metabolism. We also describe putative START-domain proteins from bacteria and unicellular protists. The majority of START domains in plants belong to a novel class of putative lipid/sterol-binding transcription factors, the HD-START family, which is conserved across the plant kingdom. HD-START proteins are confined to plants, suggesting a mechanism by which lipid/sterol ligands can directly modulate transcription in plants.

  6. IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.

    PubMed Central

    Miller, C P; McGehee, R E; Habener, J F

    1994-01-01

    We describe the cloning from a rat islet somatostatin-producing cell line of a 1.4 kb cDNA encoding a new homeoprotein, IDX-1 (islet/duodenum homeobox-1), with close sequence similarity to the Drosophila melanogaster homeobox protein Antennapedia (Antp) and the Xenopus laevis endoderm-specific homeoprotein XlHbox8. Analyses of IDX-1 mRNA and protein in rat tissues show that IDX-1 is expressed in pancreatic islets and ducts and in the duodenum. In electrophoretic mobility shift assays IDX-1 binds to three sites in the 5' flanking region of the rat somatostatin gene. In co-transfection experiments IDX-1 transactivates reporter constructs containing somatostatin promoter sequences, and mutation of the IDX-1 binding sites attenuates transactivation. Reverse transcription-polymerase chain reaction of islet RNA using degenerate amplimers for mRNAs encoding homeoproteins indicates that IDX-1 is the most abundant of 12 different Antp-like homeodomain mRNAs expressed in adult rat islets. The pattern of expression, relative abundance and transcriptional regulatory activity suggests that IDX-1 may be involved in the regulation of islet hormone genes and in cellular differentiation in the endocrine pancreas and the duodenum. Images PMID:7907546

  7. OsSLI1, a homeodomain containing transcription activator, involves abscisic acid related stress response in rice (Oryza sativa L.).

    PubMed

    Huang, Xi; Duan, Min; Liao, Jiakai; Yuan, Xi; Chen, Hui; Feng, Jiejie; Huang, Ji; Zhang, Hong-Sheng

    2014-01-01

    Homeodomain-leucine zipper type I (HD-Zip I) proteins are involved in the regulation of plant development and response to environmental stresses. In this study, OsSLI1 (Oryza sativa stress largely induced 1), encoding a member of the HD-Zip I subfamily, was isolated from rice. The expression of OsSLI1 was dramatically induced by multiple abiotic stresses and exogenous abscisic acid (ABA). In silico sequence analysis discovered several cis-acting elements including multiple ABREs (ABA-responsive element binding factors) in the upstream promoter region of OsSLI1. The OsSLI1-GFP fusion protein was localized in the nucleus of rice protoplast cells and the transcriptional activity of OsSLI1 was confirmed by the yeast hybrid system. Further, it was found that OsSLI1 expression was enhanced in an ABI5-Like1 (ABL1) deficiency rice mutant abl1 under stress conditions, suggesting that ABL1 probably negatively regulates OsSLI1 gene expression. Moreover, it was found that OsSLI1 was regulated in panicle development. Taken together, OsSLI1 may be a transcriptional activator regulating stress-responsive gene expression and panicle development in rice.

  8. Combinatorial Histone Readout by the Dual Plant Homeodomain (PHD) Fingers of Rco1 Mediates Rpd3S Chromatin Recruitment and the Maintenance of Transcriptional Fidelity.

    PubMed

    McDaniel, Stephen L; Fligor, Jennifer E; Ruan, Chun; Cui, Haochen; Bridgers, Joseph B; DiFiore, Julia V; Guo, Angela H; Li, Bing; Strahl, Brian D

    2016-07-08

    The plant homeodomain (PHD) finger is found in many chromatin-associated proteins and functions to recruit effector proteins to chromatin through its ability to bind both methylated and unmethylated histone residues. Here, we show that the dual PHD fingers of Rco1, a member of the Rpd3S histone deacetylase complex recruited to transcribing genes, operate in a combinatorial manner in targeting the Rpd3S complex to histone H3 in chromatin. Although mutations in either the first or second PHD finger allow for Rpd3S complex formation, the assembled complexes from these mutants cannot recognize nucleosomes or function to maintain chromatin structure and prevent cryptic transcriptional initiation from within transcribed regions. Taken together, our findings establish a critical role of combinatorial readout in maintaining chromatin organization and in enforcing the transcriptional fidelity of genes.

  9. Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import*

    PubMed Central

    Di Lascio, Simona; Belperio, Debora

    2016-01-01

    Heterozygous mutations of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to congenital central hypoventilation syndrome (CCHS), a neurodevelopmental disorder characterized by a failure in the autonomic control of breathing. Polyalanine expansions in the 20-residues region of the C terminus of PHOX2B are the major mutations responsible for CCHS. Elongation of the alanine stretch in PHOX2B leads to a protein with altered DNA binding, transcriptional activity, and nuclear localization and the possible formation of cytoplasmic aggregates; furthermore, the findings of various studies support the idea that CCHS is not due to a pure loss of function mechanism but also involves a dominant negative effect and/or toxic gain of function for PHOX2B mutations. Because PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant negative effects of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A using a co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers and heterodimerizes weakly with mutated proteins, exclude the direct involvement of the polyalanine tract in dimer formation, and indicate that mutated proteins retain partial ability to form heterodimers with PHOX2A. Moreover, in this study, we investigated the effects of the longest polyalanine expansions on the homeodomain-mediated nuclear import, and our data clearly show that the expanded C terminus interferes with this process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity. PMID:27129232

  10. Genome-Wide Identification and Expression Analysis of Homeodomain Leucine Zipper Subfamily IV (HDZ IV) Gene Family from Musa accuminata

    PubMed Central

    Pandey, Ashutosh; Misra, Prashant; Alok, Anshu; Kaur, Navneet; Sharma, Shivani; Lakhwani, Deepika; Asif, Mehar H.; Tiwari, Siddharth; Trivedi, Prabodh K.

    2016-01-01

    The homeodomain zipper family (HD-ZIP) of transcription factors is present only in plants and plays important role in the regulation of plant-specific processes. The subfamily IV of HDZ transcription factors (HD-ZIP IV) has primarily been implicated in the regulation of epidermal structure development. Though this gene family is present in all lineages of land plants, members of this gene family have not been identified in banana, which is one of the major staple fruit crops. In the present work, we identified 21 HDZIV encoding genes in banana by the computational analysis of banana genome resource. Our analysis suggested that these genes putatively encode proteins having all the characteristic domains of HDZIV transcription factors. The phylogenetic analysis of the banana HDZIV family genes further confirmed that after separation from a common ancestor, the banana, and poales lineages might have followed distinct evolutionary paths. Further, we conclude that segmental duplication played a major role in the evolution of banana HDZIV encoding genes. All the identified banana HDZIV genes expresses in different banana tissue, however at varying levels. The transcript levels of some of the banana HDZIV genes were also detected in banana fruit pulp, suggesting their putative role in fruit attributes. A large number of genes of this family showed modulated expression under drought and salinity stress. Taken together, the present work lays a foundation for elucidation of functional aspects of the banana HDZIV encoding genes and for their possible use in the banana improvement programs. PMID:26870050

  11. Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter.

    PubMed

    Susa, Takao; Nakayama, Michie; Kitahara, Kousuke; Kimoto, Fuyuko; Kato, Takako; Kato, Yukio

    2007-06-08

    Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.

  12. Diversity, Phylogeny and Expression Patterns of Pou and Six Homeodomain Transcription Factors in Hydrozoan Jellyfish Craspedacusta sowerbyi

    PubMed Central

    Hroudova, Miluse; Vojta, Petr; Strnad, Hynek; Krejcik, Zdenek; Ridl, Jakub; Paces, Jan; Vlcek, Cestmir; Paces, Vaclav

    2012-01-01

    Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarily conserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions. PMID:22558464

  13. A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

    PubMed Central

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

  14. Mechanism of Histone H3K4me3 Recognition by the Plant Homeodomain of Inhibitor of Growth 3*

    PubMed Central

    Kim, Sophia; Natesan, Senthil; Cornilescu, Gabriel; Carlson, Samuel; Tonelli, Marco; McClurg, Urszula L.; Binda, Olivier; Robson, Craig N.; Markley, John L.; Balaz, Stefan

    2016-01-01

    Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (KD ranging between 0.63 and 0.93 μm) and is about 200-fold stronger than with the unmodified histone H3. NMR and computational studies revealed an aromatic cage composed of Tyr-362, Ser-369, and Trp-385 that accommodate the tri-methylated side chain of H3K4. Mutational analysis confirmed the critical importance of Tyr-362 and Trp-385 in mediating the ING3PHD-H3K4me3 interaction. Finally, the biological relevance of ING3PHD-H3K4me3 binding was demonstrated by the failure of ING3PHD mutant proteins to enhance ING3-mediated DNA damage-dependent cell death. Together, our results reveal the molecular mechanism of H3K4me3 selection by the ING3PHD and suggest that this interaction is important for mediating ING3 tumor suppressive activities. PMID:27281824

  15. Characterization of PREP2, a paralog of PREP1, which defines a novel sub-family of the MEINOX TALE homeodomain transcription factors.

    PubMed

    Fognani, C; Kilstrup-Nielsen, C; Berthelsen, J; Ferretti, E; Zappavigna, V; Blasi, F

    2002-05-01

    TALE (three amino acid loop extension) homeodomain proteins include the PBC and the MEINOX sub-families. MEINOX proteins form heterodimer complexes with PBC proteins. Heterodimerization is crucial to DNA binding and for nuclear localization. PBC-MEINOX heterodimers bind DNA also in combination with HOX proteins, thereby modulating their DNA-binding specificity. TALE proteins therefore play crucial roles in multiple developmental and differentiation pathways in vivo. We report the identification and characterization of a novel human gene homologous to PREP1, called PREP2. Sequence comparisons indicate that PREP1 and PREP2 define a novel sub-family of MEINOX proteins, distinct from the MEIS sub-family. PREP2 is expressed in a variety of human adult tissues and displays a more restricted expression pattern than PREP1. PREP2 is capable of heterodimerizing with PBC proteins. Heterodimerization with PBX1 appears to be essential for nuclear localization of both PREP2 and PBX1. A comparison between the functional properties of PREP1 and PREP2 reveals that PREP2-PBX display a faster DNA-dissociation rate than PREP1-PBX heterodimers, suggesting different roles in controlling gene expression. Like PREP1, PREP2-PBX heterodimers are capable of forming ternary complexes with HOXB1. The analysis of some PREP2 in vitro properties suggests a functional diversification among PREP and between PREP and MEIS MEINOX proteins.

  16. Germ-line mutation of NKX3.1 cosegregates with hereditary prostate cancer and alters the homeodomain structure and function.

    PubMed

    Zheng, S Lilly; Ju, Jeong-ho; Chang, Bao-li; Ortner, Elizabeth; Sun, Jielin; Isaacs, Sarah D; Sun, Jishang; Wiley, Kathy E; Liu, Wennuan; Zemedkun, Micheas; Walsh, Patrick C; Ferretti, James; Gruschus, James; Isaacs, William B; Gelmann, Edward P; Xu, Jianfeng

    2006-01-01

    NKX3.1, a gene mapped to 8p21, is a member of the NK class of homeodomain proteins and is expressed primarily in the prostate. NKX3.1 exerts a growth-suppressive and differentiating effect on prostate epithelial cells. Because of its known functions and its location within a chromosomal region where evidence for prostate cancer linkage and somatic loss of heterozygosity is found, we hypothesize that sequence variants in the NKX3.1 gene increase prostate cancer risk. To address this, we first resequenced the NKX3.1 gene in 159 probands of hereditary prostate cancer families recruited at Johns Hopkins Hospital; each family has at least three first-degree relatives affected with prostate cancer. Twenty-one germ-line variants were identified in this analysis, including one previously described common nonsynonymous change (R52C), two novel rare nonsynonymous changes (A17T and T164A), and a novel common 18-bp deletion in the promoter. Overall, the germ-line variants were significantly linked to prostate cancer, with a peak heterogeneity logarithm of odds of 2.04 (P = 0.002) at the NKX3.1 gene. The rare nonsynonymous change, T164A, located in the homeobox domain of the gene, segregated with prostate cancer in a family with three affected brothers and one unaffected brother. Importantly, nuclear magnetic resonance solution structure analysis and circular dichroism studies showed this specific mutation to affect the stability of the homeodomain of the NKX3.1 protein and decreased binding to its cognate DNA recognition sequence. These results suggest that germ-line sequence variants in NKX3.1 may play a role in susceptibility to hereditary prostate cancer and underscore a role for NKX3.1 as a prostate cancer gatekeeper.

  17. New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

    PubMed Central

    Zucchelli, Chiara; Ferrari, Elena; Blasi, Francesco; Musco, Giovanna; Bruckmann, Chiara

    2017-01-01

    PREP1 and PBX1 are homeodomain (HD) transcription factors that play crucial roles in embryonic development. Here, we present the first biophysical characterization of a PREP1 HD, and the NMR spectroscopic study of its DNA binding pocket. The data show that residues flanking the HD participate in DNA binding. The kinetic parameters for DNA binding of individual PREP1 and PBX1 HDs, and of their combination, show that isolated PREP1 and PBX1 HDs bind to DNA in a cooperative manner. A novel PREP1 motif, flanking the HD at the C-terminus, is required for cooperativity. PMID:28094776

  18. Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

    PubMed

    Busser, Brian W; Gisselbrecht, Stephen S; Shokri, Leila; Tansey, Terese R; Gamble, Caitlin E; Bulyk, Martha L; Michelson, Alan M

    2013-01-01

    Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I-HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks

  19. Integrative genomic and transcriptomic analysis for pinpointing recurrent alterations of plant homeodomain genes and their clinical significance in breast cancer.

    PubMed

    Yu, Huimei; Jiang, Yuanyuan; Liu, Lanxin; Shan, Wenqi; Chu, Xiaofang; Yang, Zhe; Yang, Zeng-Quan

    2017-02-21

    A wide range of the epigenetic effectors that regulate chromatin modification, gene expression, genomic stability, and DNA repair contain structurally conserved domains called plant homeodomain (PHD) fingers. Alternations of several PHD finger-containing proteins (PHFs) due to genomic amplification, mutations, deletions, and translocations have been linked directly to various types of cancer. However, little is known about the genomic landscape and the clinical significance of PHFs in breast cancer. Hence, we performed a large-scale genomic and transcriptomic analysis of 98 PHF genes in breast cancer using TCGA and METABRIC datasets and correlated the recurrent alterations with clinicopathological features and survival of patients. Different subtypes of breast cancer had different patterns of copy number and expression for each PHF. We identified a subset of PHF genes that was recurrently altered with high prevalence, including PYGO2 (pygopus family PHD finger 2), ZMYND8 (zinc finger, MYND-type containing 8), ASXL1 (additional sex combs like 1) and CHD3 (chromodomain helicase DNA binding protein 3). Copy number increase and overexpression of ZMYND8 were more prevalent in Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. ZMYND8 was also involved in a positive feedback circuit of the estrogen receptor (ER) pathway, and the expression of ZMYND8 was repressed by the bromodomain and extra terminal (BET) inhibitor in breast cancer. Our findings suggest a promising avenue for future research-to focus on a subset of PHFs to better understand the molecular mechanisms and to identify therapeutic targets in breast cancer.

  20. Integrative genomic and transcriptomic analysis for pinpointing recurrent alterations of plant homeodomain genes and their clinical significance in breast cancer

    PubMed Central

    Yu, Huimei; Jiang, Yuanyuan; Liu, Lanxin; Shan, Wenqi; Chu, Xiaofang; Yang, Zhe; Yang, Zeng-Quan

    2017-01-01

    A wide range of the epigenetic effectors that regulate chromatin modification, gene expression, genomic stability, and DNA repair contain structurally conserved domains called plant homeodomain (PHD) fingers. Alternations of several PHD finger-containing proteins (PHFs) due to genomic amplification, mutations, deletions, and translocations have been linked directly to various types of cancer. However, little is known about the genomic landscape and the clinical significance of PHFs in breast cancer. Hence, we performed a large-scale genomic and transcriptomic analysis of 98 PHF genes in breast cancer using TCGA and METABRIC datasets and correlated the recurrent alterations with clinicopathological features and survival of patients. Different subtypes of breast cancer had different patterns of copy number and expression for each PHF. We identified a subset of PHF genes that was recurrently altered with high prevalence, including PYGO2 (pygopus family PHD finger 2), ZMYND8 (zinc finger, MYND-type containing 8), ASXL1 (additional sex combs like 1) and CHD3 (chromodomain helicase DNA binding protein 3). Copy number increase and overexpression of ZMYND8 were more prevalent in Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. ZMYND8 was also involved in a positive feedback circuit of the estrogen receptor (ER) pathway, and the expression of ZMYND8 was repressed by the bromodomain and extra terminal (BET) inhibitor in breast cancer. Our findings suggest a promising avenue for future research—to focus on a subset of PHFs to better understand the molecular mechanisms and to identify therapeutic targets in breast cancer. PMID:28055972

  1. Foxp2 inhibits Nkx2.1-mediated transcription of SP-C via interactions with the Nkx2.1 homeodomain.

    PubMed

    Zhou, Beiyun; Zhong, Qian; Minoo, Parviz; Li, Changgong; Ann, David K; Frenkel, Baruch; Morrisey, Edward E; Crandall, Edward D; Borok, Zea

    2008-06-01

    The transcription factor (TF) Foxp2 has been shown to partially repress surfactant protein C (SP-C) transcription, presumably through interaction of an independent repressor domain with a conserved Foxp2 consensus site in the SP-C promoter. We explored the role of interactions between Foxp2 and the homeodomain TF Nkx2.1 that may contribute to the marked reduction in SP-C expression accompanying phenotypic transition of alveolar epithelial type II (AT2) to type I (AT1) cells. Foxp2 dose-dependently inhibited Nkx2.1-mediated activation of SP-C in MLE-15 cells. While electrophoretic mobility shift assays and chromatin immunoprecipitations revealed an interaction between Foxp2 and the conserved consensus motif in the SP-C promoter, Nkx2.1-mediated activation of the 318-bp proximal SP-C promoter (which lacks a Foxp2 consensus) was attenuated by increasing amounts of Foxp2. Co-immunoprecipitation and mammalian two-hybrid assays confirmed a physical interaction between Nkx2.1 and Foxp2 mediated through the Nkx2.1 homeodomain. Formation of an Nkx2.1 complex with an SP-C oligonucleotide was inhibited dose-dependently by recombinant Foxp2. These findings demonstrate that direct interaction between Foxp2 and Nkx2.1 inhibits Nkx2.1 DNA-binding and transcriptional activity and suggest a mechanism for down-regulation of SP-C (and probably other AT2 cell genes) during transition of AT2 cells to an AT1 cell phenotype.

  2. The Homeodomain Transcription Factors Antennapedia and POU-M2 Regulate the Transcription of the Steroidogenic Enzyme Gene Phantom in the Silkworm*

    PubMed Central

    Meng, Meng; Cheng, Dao-jun; Peng, Jian; Qian, Wen-liang; Li, Jia-rui; Dai, Dan-dan; Zhang, Tian-lei; Xia, Qing-you

    2015-01-01

    The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis. PMID:26253172

  3. Characterization and mutation analysis of goosecoid-like (GSCL), a homeodomain-containing gene that maps to the critical region for VCFS/DGS on 22q11.

    PubMed

    Funke, B; Saint-Jore, B; Puech, A; Sirotkin, H; Edelmann, L; Carlson, C; Raft, S; Pandita, R K; Kucherlapati, R; Skoultchi, A; Morrow, B E

    1997-12-15

    Velocardiofacial syndrome (VCFS) is a developmental disorder characterized by conotruncal heart defects, craniofacial anomalies, and learning disabilities. VCFS is phenotypically related to DiGeorge syndrome (DGS) and both syndromes are associated with hemizygous 22q11 deletions. Because many of the tissues and structures affected in VCFS/DGS derive from the pharyngeal arches of the developing embryo, it is believed that haploinsufficiency of a gene(s) involved in embryonic development may be responsible for its etiology. A homeodomain-containing gene, Goosecoidlike (GSCL), has been recently described, and it resides in the critical region for VCFS/DGS on 22q11. GSCL is related to the Goosecoid gene (GSC) in both sequence of the homeodomain and genomic organization. Gsc in the mouse is expressed during early and midembryogenesis and is required for craniofacial rib, and limb development. The chick homolog of GSCL, termed GSX, is expressed during early chick embryogenesis. We detected GSCL expression in human embryos and biphasic expression in mouse embryos. It is possible that the vertebrate GSCL gene is also required for embryonic development. Due to its location in the critical region on 22q11, GSCL is an excellent candidate gene for VCFS/DGS. The vertebrate GSC protein has the same DNA binding specificity as the Drosophila morphogen, bicoid. Upon examination of the putative GSCL promoter, we found three sequence elements with an exact match to the reverse complement of the bicoid DNA recognition motif, suggesting that GSC, or possibly GSCL itself, regulates the transcription of GSCL. Sequence analysis of the putative promoter and the coding region of GSCL was performed on the DNA template from 17 VCFS patients who did not have a detectable 22q11 deletion to identify mutations. We did not detect a mutation in this set of VCFS patients. A polymorphism was detected in codon 47 of exon 1.

  4. Molecular cloning of LIM homeodomain transcription factor Lhx2 as a transcription factor of porcine follicle-stimulating hormone beta subunit (FSHβ) gene.

    PubMed

    Kato, Takako; Ishikawa, Akio; Yoshida, Saishu; Sano, Yoshiya; Kitahara, Kousuke; Nakayama, Michie; Susa, Takao; Kato, Yukio

    2012-01-01

    We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone β subunit gene (Fshβ) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LβT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LβT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshβ, it is possible that LHX2 controls the synthesis of FSH at the transcription level.

  5. Intact structure of EGAM1 homeoproteins and basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C contribute to their nuclear localization in mouse embryonic stem cells.

    PubMed

    Sato, Sho; Morita, Sanae; Iha, Momoe; Mori, Yuki; Sugawara, Saiko; Kasuga, Kano; Kojima, Ikuo; Ozaki, Noriaki; Muraguchi, Hajime; Okano, Keiju; Iwashita, Jun; Murata, Jun; Hosaka, Masahiro; Kobayashi, Masayuki

    2013-08-01

    Recently, we identified the structurally related homeoproteins EGAM1, EGAM1N, and EGAM1C in both preimplantation mouse embryos and mouse embryonic stem (ES) cells. These EGAM1 homeoproteins act as positive or negative regulators of differentiation and cell growth in mouse ES cells, such that these proteins are considered transcriptional regulators. In this study, we investigated their nuclear localization and identified the amino acid residues crucial for the nuclear translocation of EGAM1 and EGAM1C. When expressed exogenously in pluripotent ES cells and somatic NIH3T3 cells, all EGAM1 homeoproteins localized to the nucleus. Analysis using the web-based tool PSORTII predicted a potential nuclear localization signal (NLS) motif, RKDLIRSWFITQRHR, in the homeodomain shared by EGAM1 and EGAM1C. The introduction of mutations, such as mutations from K or R, both basic amino acid residues, to A, in this potential NLS resulted in significant impairment of the nuclear localization of both EGAM1 and EGAM1C. In contrast, GFP fusion proteins of all the full-length EGAM1 homeoproteins failed to localize to the nucleus. These results, when taken together, suggest that basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C and the intact structures of the EGAM1 homeoproteins contribute, at least in part, to the nuclear localization of these proteins in mouse ES cells. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks

    PubMed Central

    Su, Wen-Pin; Hsu, Sen-Huei; Chia, Li-Chiao; Lin, Jui-Yang; Chang, Song-Bin; Jiang, Zong-da; Lin, Yi-Ju; Shih, Min-Yu; Chen, Yi-Cheng; Chang, Mau-Sun; Yang, Wen-Bin; Hung, Jan-Jong; Hung, Po-Cheng; Wu, Wei-Sheng; Myung, Kyungjae; Liaw, Hungjiun

    2016-01-01

    DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site. PMID:26564157

  7. KNOX Lost the OX: The Arabidopsis KNATM Gene Defines a Novel Class of KNOX Transcriptional Regulators Missing the Homeodomain

    USDA-ARS?s Scientific Manuscript database

    In this study, we identify and characterize the Arabidopsis thaliana KNATM gene, which encodes a MEINOX domain but not a homeodomain. Phylogenetic analysis of the KNOX family places KNATM in a new class and shows conservation in dicotyledons. We demonstrate that KNATM selectively interacts with Arab...

  8. The Populus ARBORKNOX1 homeodomain transcription factor regulates woody growth through binding to evolutionarily conserved target genes of diverse function

    Treesearch

    Lijun Liu; Matthew S. Zinkgraf; H. Earl Petzold; Eric P. Beers; Vladimir Filkov; Andrew Groover

    2014-01-01

    The class I KNOX homeodomain transcription factor ARBORKNOX1 (ARK1) is a key regulator of vascular cambium maintenance and cell differentiation in Populus. Currently, basic information is lacking concerning the distribution, functional characteristics, and evolution of ARK1 binding in the Populus genome.

  9. The Pax6b homeodomain is dispensable for pancreatic endocrine cell differentiation in zebrafish.

    PubMed

    Verbruggen, Vincianne; Ek, Olivier; Georlette, Daphné; Delporte, François; Von Berg, Virginie; Detry, Nathalie; Biemar, Frédéric; Coutinho, Pedro; Martial, Joseph A; Voz, Marianne L; Manfroid, Isabelle; Peers, Bernard

    2010-04-30

    Pax6 is a well conserved transcription factor that contains two DNA-binding domains, a paired domain and a homeodomain, and plays a key role in the development of eye, brain, and pancreas in vertebrates. The recent identification of the zebrafish sunrise mutant, harboring a mutation in the pax6b homeobox and presenting eye abnormalities but no obvious pancreatic defects, raised a question about the role of pax6b in zebrafish pancreas. We show here that pax6b does play an essential role in pancreatic endocrine cell differentiation, as revealed by the phenotype of a novel zebrafish pax6b null mutant and of embryos injected with pax6b morpholinos. Pax6b-depleted embryos have almost no beta cells, a strongly reduced number of delta cells, and a significant increase of epsilon cells. Through the use of various morpholinos targeting intron-exon junctions, pax6b RNA splicing was perturbed at several sites, leading either to retention of intronic sequences or to deletion of exonic sequences in the pax6b transcript. By this strategy, we show that deletion of the Pax6b homeodomain in zebrafish embryos does not disturb pancreas development, whereas lens formation is strongly affected. These data thus provide the explanation for the lack of pancreatic defects in the sunrise pax6b mutants. In addition, partial reduction of Pax6b function in zebrafish embryos performed by injection of small amounts of pax6b morpholinos caused a clear rise in alpha cell number and in glucagon expression, emphasizing the importance of the fine tuning of the Pax6b level to its biological activity.

  10. The Pax6b Homeodomain Is Dispensable for Pancreatic Endocrine Cell Differentiation in Zebrafish*

    PubMed Central

    Verbruggen, Vincianne; Ek, Olivier; Georlette, Daphné; Delporte, François; Von Berg, Virginie; Detry, Nathalie; Biemar, Frédéric; Coutinho, Pedro; Martial, Joseph A.; Voz, Marianne L.; Manfroid, Isabelle; Peers, Bernard

    2010-01-01

    Pax6 is a well conserved transcription factor that contains two DNA-binding domains, a paired domain and a homeodomain, and plays a key role in the development of eye, brain, and pancreas in vertebrates. The recent identification of the zebrafish sunrise mutant, harboring a mutation in the pax6b homeobox and presenting eye abnormalities but no obvious pancreatic defects, raised a question about the role of pax6b in zebrafish pancreas. We show here that pax6b does play an essential role in pancreatic endocrine cell differentiation, as revealed by the phenotype of a novel zebrafish pax6b null mutant and of embryos injected with pax6b morpholinos. Pax6b-depleted embryos have almost no β cells, a strongly reduced number of δ cells, and a significant increase of ϵ cells. Through the use of various morpholinos targeting intron-exon junctions, pax6b RNA splicing was perturbed at several sites, leading either to retention of intronic sequences or to deletion of exonic sequences in the pax6b transcript. By this strategy, we show that deletion of the Pax6b homeodomain in zebrafish embryos does not disturb pancreas development, whereas lens formation is strongly affected. These data thus provide the explanation for the lack of pancreatic defects in the sunrise pax6b mutants. In addition, partial reduction of Pax6b function in zebrafish embryos performed by injection of small amounts of pax6b morpholinos caused a clear rise in α cell number and in glucagon expression, emphasizing the importance of the fine tuning of the Pax6b level to its biological activity. PMID:20177065

  11. A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members' expression in tomato.

    PubMed

    Zhang, Zhenzhu; Chen, Xiuling; Guan, Xin; Liu, Yang; Chen, Hongyu; Wang, Tingting; Mouekouba, Liana Dalcantara Ongouya; Li, Jingfu; Wang, Aoxue

    2014-01-01

    Homeodomain-leucine zipper (HD-Zip) proteins are a kind of transcriptional factors that play a vital role in plant growth and development. However, no detailed information of HD-Zip family in tomato has been reported till now. In this study, 51 HD-Zip genes (SlHZ01-51) in this family were identified and categorized into 4 classes by exon-intron and protein structure in tomato (Solanum lycopersicum) genome. The synthetical phylogenetic tree of tomato, Arabidopsis and rice HD-Zip genes were established for an insight into their evolutionary relationships and putative functions. The results showed that the contribution of segmental duplication was larger than that of tandem duplication for expansion and evolution of genes in this family of tomato. The expression profile results under abiotic stress suggested that all SlHZ I genes were responsive to cold stress. This study will provide a clue for the further investigation of functional identification and the role of tomato HD-Zip I subfamily in plant cold stress responses and developmental events.

  12. The homeodomain transcription factor TaHDZipI-2 from wheat regulates frost tolerance, flowering time and spike development in transgenic barley.

    PubMed

    Kovalchuk, Nataliya; Chew, William; Sornaraj, Pradeep; Borisjuk, Nikolai; Yang, Nannan; Singh, Rohan; Bazanova, Natalia; Shavrukov, Yuri; Guendel, Andre; Munz, Eberhard; Borisjuk, Ljudmilla; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2016-07-01

    Homeodomain leucine zipper class I (HD-Zip I) transcription factors (TFs) play key roles in the regulation of plant growth and development under stresses. Functions of the TaHDZipI-2 gene isolated from the endosperm of developing wheat grain were revealed. Molecular characterization of TaHDZipI-2 protein included studies of its dimerisation, protein-DNA interactions and gene activation properties using pull-down assays, in-yeast methods and transient expression assays in wheat cells. The analysis of TaHDZipI-2 gene functions was performed using transgenic barley plants. It included comparison of developmental phenotypes, yield components, grain quality, frost tolerance and the levels of expression of potential target genes in transgenic and control plants. Transgenic TaHDZipI-2 lines showed characteristic phenotypic features that included reduced growth rates, reduced biomass, early flowering, light-coloured leaves and narrowly elongated spikes. Transgenic lines produced 25-40% more seeds per spike than control plants, but with 50-60% smaller grain size. In vivo lipid imaging exposed changes in the distribution of lipids between the embryo and endosperm in transgenic seeds. Transgenic lines were significantly more tolerant to frost than control plants. Our data suggest the role of TaHDZipI-2 in controlling several key processes underlying frost tolerance, transition to flowering and spike development.

  13. Role of Homeodomain leucine zipper (HD-Zip) IV transcription factors in plant development and plant protection from deleterious environmental factors.

    PubMed

    Chew, William; Hrmova, Maria; Lopato, Sergiy

    2013-04-12

    Homeobox genes comprise an important group of genes that are responsible for regulation of developmental processes. These genes determine cell differentiation and cell fate in all eukaryotic organisms, starting from the early stages of embryo development. Homeodomain leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom. Members of the HD-Zip IV subfamily have a complex domain topology and can bind several cis-elements with overlapping sequences. Many of the reported HD-Zip IV genes were shown to be specifically or preferentially expressed in plant epidermal or sub-epidermal cells. HD-Zip IV TFs were found to be associated with differentiation and maintenance of outer cell layers, and regulation of lipid biosynthesis and transport. Insights about the role of these proteins in plant cuticle formation, and hence their possible involvement in plant protection from pathogens and abiotic stresses has just started to emerge. These roles make HD-Zip IV proteins an attractive tool for genetic engineering of crop plants. To this end, there is a need for in-depth studies to further clarify the function of each HD-Zip IV subfamily member in commercially important plant species.

  14. Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

    SciTech Connect

    Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C.; Kolatkar, Prasanna R.

    2010-02-08

    The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

  15. Bell-like homeodomain selectively regulates the high-irradiance response of phytochrome A.

    PubMed

    Staneloni, Roberto J; Rodriguez-Batiller, María José; Legisa, Danilo; Scarpin, María R; Agalou, Adamantia; Cerdán, Pablo D; Meijer, Annemarie H; Ouwerkerk, Pieter B F; Casal, Jorge J

    2009-08-11

    Plant responses mediated by phytochrome A display a first phase saturated by transient light signals and a second phase requiring sustained excitation with far-red light (FR). These discrete outcomes, respectively so-called very-low-fluence response (VLFR) and high-irradiance response (HIR), are appropriate in different environmental and developmental contexts but the mechanisms that regulate the switch remain unexplored. Promoter analysis of a light-responsive target gene revealed a motif necessary for HIR but not for VLFR. This motif is required for binding of the Bell-like homeodomain 1 (BLH1) to the promoter in in vitro and in yeast 1-hybrid experiments. Promoter substitutions that increased BLH1 binding also enhanced HIR. blh1 mutants showed reduced responses to continuous FR and to deep canopy shadelight, but they retained normal responses to pulsed FR or red light and unfiltered sunlight. BLH1 enhanced BLH1 expression and its promotion by FR. We conclude that BLH1 specifically regulates HIR and not VLFR of phytochrome A.

  16. Bell-like homeodomain selectively regulates the high-irradiance response of phytochrome A

    PubMed Central

    Staneloni, Roberto J.; Rodriguez-Batiller, María José; Legisa, Danilo; Scarpin, María R.; Agalou, Adamantia; Cerdán, Pablo D.; Meijer, Annemarie H.; Ouwerkerk, Pieter B. F.; Casal, Jorge J.

    2009-01-01

    Plant responses mediated by phytochrome A display a first phase saturated by transient light signals and a second phase requiring sustained excitation with far-red light (FR). These discrete outcomes, respectively so-called very-low-fluence response (VLFR) and high-irradiance response (HIR), are appropriate in different environmental and developmental contexts but the mechanisms that regulate the switch remain unexplored. Promoter analysis of a light-responsive target gene revealed a motif necessary for HIR but not for VLFR. This motif is required for binding of the Bell-like homeodomain 1 (BLH1) to the promoter in in vitro and in yeast 1-hybrid experiments. Promoter substitutions that increased BLH1 binding also enhanced HIR. blh1 mutants showed reduced responses to continuous FR and to deep canopy shadelight, but they retained normal responses to pulsed FR or red light and unfiltered sunlight. BLH1 enhanced BLH1 expression and its promotion by FR. We conclude that BLH1 specifically regulates HIR and not VLFR of phytochrome A. PMID:19666535

  17. Hierarchical Interactions of Homeodomain and Forkhead Transcription Factors in Regulating Odontogenic Gene Expression*

    PubMed Central

    Venugopalan, Shankar R.; Li, Xiao; Amen, Melanie A.; Florez, Sergio; Gutierrez, Diana; Cao, Huojun; Wang, Jianbo; Amendt, Brad A.

    2011-01-01

    FoxJ1 is a forkhead transcription factor expressed in multiple tissues during development and a major regulator of cilia development. FoxJ1−/− mice present with defects in odontogenesis, and we correlate these defects to hierarchical interactions between homeodomain factors Pitx2 and Dlx2 with FoxJ1 in regulating their expression through direct physical interactions. Chromatin immunoprecipitation assays reveal endogenous Pitx2 and Dlx2 binding to the Dlx2 promoter and Dlx2 binding to the FoxJ1 promoter as well as Dlx2 and FoxJ1 binding to the amelogenin promoter. PITX2 activation of the Dlx2 promoter is attenuated by a direct Dlx2 physical interaction with PITX2. Dlx2 autoregulates its promoter, and Dlx2 transcriptionally activates the downstream gene FoxJ1. Dlx2 and FoxJ1 physically interact and synergistically regulate both Dlx2 and FoxJ1 promoters. Dlx2 and FoxJ1 also activate the amelogenin promoter, and amelogenin is required for enamel formation and late stage tooth development. FoxJ1−/− mice maxillary and mandibular incisors are reduced in length and width and have reduced amelogenin expression. FoxJ1−/− mice show a reduced and defective ameloblast layer, revealing a biological effect of these transcription factor hierarchies during tooth morphogenesis. These transcriptional mechanisms may contribute to other developmental processes such as neuronal, pituitary, and heart development. PMID:21504905

  18. Establishing a Framework for the Ad/Abaxial Regulatory Network of Arabidopsis: Ascertaining Targets of Class III HOMEODOMAIN LEUCINE ZIPPER and KANADI Regulation[W

    PubMed Central

    Reinhart, Brenda J.; Liu, Tie; Newell, Nicole R.; Magnani, Enrico; Huang, Tengbo; Kerstetter, Randall; Michaels, Scott; Barton, M. Kathryn

    2013-01-01

    The broadly conserved Class III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator–target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as INDETERMINATE DOMAIN4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development. PMID:24076978

  19. Establishing a framework for the Ad/abaxial regulatory network of Arabidopsis: ascertaining targets of class III homeodomain leucine zipper and KANADI regulation.

    PubMed

    Reinhart, Brenda J; Liu, Tie; Newell, Nicole R; Magnani, Enrico; Huang, Tengbo; Kerstetter, Randall; Michaels, Scott; Barton, M Kathryn

    2013-09-01

    The broadly conserved Class III homeodomain leucine zipper (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator-target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as indeterminate domain4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development.

  20. Molecular interactions of the γ-clade homeodomain-leucine zipper class I transcription factors during the wheat response to water deficit.

    PubMed

    Harris, John C; Sornaraj, Pradeep; Taylor, Mathew; Bazanova, Natalia; Baumann, Ute; Lovell, Ben; Langridge, Peter; Lopato, Sergiy; Hrmova, Maria

    2016-03-01

    The γ-clade of class I homeodomain-leucine zipper (HD-Zip I) transcription factors (TFs) constitute members which play a role in adapting plant growth to conditions of water deficit. Given the importance of wheat (Triticum aestivum L.) as a global food crop and the impact of water deficit upon grain yield, we focused on functional aspects of wheat drought responsive HD-Zip I TFs. While the wheat γ-clade HD-Zip I TFs share significant sequence similarities with homologous genes from other plants, the clade-specific features in transcriptional response to abiotic stress were detected. We demonstrate that wheat TaHDZipI-3, TaHDZipI-4, and TaHDZipI-5 genes respond differentially to a variety of abiotic stresses, and that proteins encoded by these genes exhibit pronounced differences in oligomerisation, strength of DNA binding, and trans-activation of an artificial promoter. Three-dimensional molecular modelling of the protein-DNA interface was conducted to address the ambiguity at the central nucleotide in the pseudo-palindromic cis-element CAATNATTG that is recognised by all three HD-Zip I proteins. The co-expression of these genes in the same plant tissues together with the ability of HD-Zip I TFs of the γ-clade to hetero-dimerise suggests a role in the regulatory mechanisms of HD-Zip I dependent transcription. Our findings highlight the complexity of TF networks involved in plant responses to water deficit. A better understanding of the molecular complexity at the protein level during crop responses to drought will enable adoption of efficient strategies for production of cereal plants with enhanced drought tolerance.

  1. Plant homeodomain-leucine zipper I transcription factors exhibit different functional AHA motifs that selectively interact with TBP or/and TFIIB.

    PubMed

    Capella, Matías; Ré, Delfina A; Arce, Agustín L; Chan, Raquel L

    2014-06-01

    Different members of the HD-Zip I family of transcription factors exhibit differential AHA-like activation motifs, able to interact with proteins of the basal transcriptional machinery. Homeodomain-leucine zipper proteins are transcription factors unique to plants, classified in four subfamilies. Subfamily I members have been mainly associated to abiotic stress responses. Several ones have been characterized using knockout or overexpressors plants, indicating that they take part in different signal transduction pathways even when their expression patterns are similar and they bind the same DNA sequence. A bioinformatic analysis has revealed the existence of conserved motifs outside the HD-Zip domain, including transactivation AHA motifs. Here, we demonstrate that these putative activation motifs are functional. Four members of the Arabidopsis family were chosen: AtHB1, AtHB7, AtHB12 and AtHB13. All of them exhibited activation activity in yeast and in plants but with different degrees. The protein segment necessary for such activation was different for these four transcription factors as well as the role of the tryptophans they present. When interaction with components of the basal transcription machinery was tested, AtHB1 was able to interact with TBP, AtHB12 interacted with TFIIB, AtHB7 interacted with both, TBP and TFIIB while AtHB13 showed weak interactions with any of them, in yeast two-hybrid as well as in pull-down assays. Transient transformation of Arabidopsis seedlings confirmed the activation capacity and specificity of these transcription factors and showed some differences with the results obtained in yeast. In conclusion, the differential activation functionality of these transcription factors adds an important level of functional divergence of these proteins, and together with their expression patterns, these differences could explain, at least in part, their functional divergence.

  2. Epidermal cell differentiation in cotton mediated by the homeodomain leucine zipper gene, GhHD-1.

    PubMed

    Walford, Sally-Ann; Wu, Yingru; Llewellyn, Danny J; Dennis, Elizabeth S

    2012-08-01

    Gossypium hirsutum L. (cotton) fibres are specialized trichomes a few centimetres in length that grow from the seed coat. Few genes directly involved in the differentiation of these epidermal cells have been identified. These include GhMYB25-like and GhMYB25, two related MYB transcription factors that regulate fibre cell initiation and expansion. We have also identified a putative homeodomain leucine zipper (HD-ZIP) transcription factor, GhHD-1, expressed in trichomes and early fibres that might play a role in cotton fibre initiation. Here, we characterize GhHD-1 homoeologues from tetraploid G. hirsutum and show, using reporter constructs and quantitative real-time PCR (qRT-PCR), that they are expressed predominantly in epidermal tissues during early fibre development, and in other tissues bearing epidermal trichomes. Silencing of GhHD-1 reduced trichome formation and delayed the timing of fibre initiation. Constitutive overexpression of GhHD-1 increased the number of fibres initiating on the seed, but did not affect leaf trichomes. Expression of GhHD-1 in cotton silenced for different fibre MYBs suggest that in ovules it acts downstream of GhMYB25-like, but is unaffected in GhMYB25- or GhMYB109-silenced plants. Microarray analysis of silencing and overexpression lines of GhHD-1 indicated that it potentially regulates the levels of ethylene and reactive oxidation species (ROS) through a WRKY transcription factor and calcium-signalling pathway genes to activate downstream genes necessary for cell expansion and elongation. © 2012 CSIRO. The Plant Journal © 2012 Blackwell Publishing Ltd.

  3. Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development

    PubMed Central

    Verzi, Michael P.; Stanfel, Monique N.; Moses, Kelvin A.; Kim, Byeong-Moo; Zhang, Yan; Schwartz, Robert J.; Shivdasani, Ramesh A.; Zimmer, Warren E.

    2010-01-01

    Background & Aims Expansion and patterning of the endoderm generate a highly ordered, multi-organ digestive system in vertebrate animals. Among distal foregut derivatives, the gastric corpus, antrum, pylorus and duodenum are distinct structures with sharp boundaries. Some homeodomain transcription factors expressed in gut mesenchyme convey positional information required for anterior-posterior patterning of the digestive tract. Barx1, in particular, controls stomach differentiation and morphogenesis. The NK homeobox gene Bapx1 (Nkx3-2) has an established role in skeletal development but its function in the mammalian gut is less clear. Methods We generated a Bapx1Cre knock-in allele to fate map Bapx1-expressing cells and evaluate its function in gastrointestinal development. Results Bapx1-expressing cells populate the gut mesenchyme with a rostral boundary in the hindstomach, near the junction of the gastric corpus and antrum. Smooth muscle differentiation and distribution of early regional markers are ostensibly normal in Bapx1Cre/Cre gut, but there are distinctive morphologic abnormalities near this rostral Bapx1 domain: the antral segment of the stomach is markedly shortened and the pyloric constriction is lost. Comparison of expression domains and examination of stomach phenotypes in single and compound Barx1 and Bapx1 mutant mice suggest a hierarchy between these two factors; Bapx1 expression is lost in the absence of Barx1. Conclusions This study reveals the non-redundant requirement for Bapx1 in distal stomach development, places it within a Barx1-dependent pathway, and illustrates the pervasive influence of gut mesenchyme homeobox genes on endoderm differentiation and digestive organogenesis. PMID:19208343

  4. The homeodomain complement of the ctenophore Mnemiopsis leidyi suggests that Ctenophora and Porifera diverged prior to the ParaHoxozoa

    PubMed Central

    2010-01-01

    Background The much-debated phylogenetic relationships of the five early branching metazoan lineages (Bilateria, Cnidaria, Ctenophora, Placozoa and Porifera) are of fundamental importance in piecing together events that occurred early in animal evolution. Comparisons of gene content between organismal lineages have been identified as a potentially useful methodology for phylogenetic reconstruction. However, these comparisons require complete genomes that, until now, did not exist for the ctenophore lineage. The homeobox superfamily of genes is particularly suited for these kinds of gene content comparisons, since it is large, diverse, and features a highly conserved domain. Results We have used a next-generation sequencing approach to generate a high-quality rough draft of the genome of the ctenophore Mnemiopsis leidyi and subsequently identified a set of 76 homeobox-containing genes from this draft. We phylogenetically categorized this set into established gene families and classes and then compared this set to the homeodomain repertoire of species from the other four early branching metazoan lineages. We have identified several important classes and subclasses of homeodomains that appear to be absent from Mnemiopsis and from the poriferan Amphimedon queenslandica. We have also determined that, based on lineage-specific paralog retention and average branch lengths, it is unlikely that these missing classes and subclasses are due to extensive gene loss or unusually high rates of evolution in Mnemiopsis. Conclusions This paper provides a first glimpse of the first sequenced ctenophore genome. We have characterized the full complement of Mnemiopsis homeodomains from this species and have compared them to species from other early branching lineages. Our results suggest that Porifera and Ctenophora were the first two extant lineages to diverge from the rest of animals. Based on this analysis, we also propose a new name - ParaHoxozoa - for the remaining group that

  5. Identification and mapping of Tril, a homeodomain-leucine zipper gene involved in multicellular trichome initiation in Cucumis sativus.

    PubMed

    Wang, Yun-Li; Nie, Jing-tao; Chen, Hui-Ming; Guo, Chun-li; Pan, Jian; He, Huan-Le; Pan, Jun-Song; Cai, Run

    2016-02-01

    Using map-based cloning of Tril gene, we identified a homeodomain-leucine zipper gene involved in the initiation of multicellular trichomes (including the spines of fruit) in cucumber. Fruit spines are a special type of trichome that impacts the quality and appearance of cucumber (Cucumis sativus L.) fruit. Scanning electron microscopy revealed that the trichome-less (tril) mutant originating from European greenhouse cucumber has a completely glabrous phenotype on cotyledons, hypocotyls, young leaves, fruits, and fruit stalks. Genetic analysis revealed that tril was inherited as a recessive allele at a single locus. Using 1058 F2 individuals derived from a cross between cucumber tril mutant CGN19839 and the micro-trichome (mict) mutant 06-2, tril was mapped to chromosome 6, and narrowed down to a 37.4 kb genomic region which carries seven predicted genes. Genetic and molecular analyses revealed that gene Cucsa.045360 is a possible candidate gene for the differentiation of epidermal cells to trichomes. It is a member of the class IV homeodomain-leucine zipper (HD-Zip IV) family and encodes homeodomain and START domain, sharing 66.7% predicted amino acid sequence identity to PROTODERMAL FACTOR2 (PDF2) and 35.0% to GLABRA2 (GL2) of Arabidopsis. The homeobox domain had changed amino acid sequence because of an insertion in tril mutant. The results of genetic analysis and transcriptome profiling indicated that the Tril gene had an epistatic effect on the Mict gene in trichome development. Phenotypes of the tril mutant such as glabrous fruits and female flowers at every node could be used in developing new cultivars.

  6. The homeodomain complement of the ctenophore Mnemiopsis leidyi suggests that Ctenophora and Porifera diverged prior to the ParaHoxozoa.

    PubMed

    Ryan, Joseph F; Pang, Kevin; Mullikin, James C; Martindale, Mark Q; Baxevanis, Andreas D

    2010-10-04

    The much-debated phylogenetic relationships of the five early branching metazoan lineages (Bilateria, Cnidaria, Ctenophora, Placozoa and Porifera) are of fundamental importance in piecing together events that occurred early in animal evolution. Comparisons of gene content between organismal lineages have been identified as a potentially useful methodology for phylogenetic reconstruction. However, these comparisons require complete genomes that, until now, did not exist for the ctenophore lineage. The homeobox superfamily of genes is particularly suited for these kinds of gene content comparisons, since it is large, diverse, and features a highly conserved domain. We have used a next-generation sequencing approach to generate a high-quality rough draft of the genome of the ctenophore Mnemiopsis leidyi and subsequently identified a set of 76 homeobox-containing genes from this draft. We phylogenetically categorized this set into established gene families and classes and then compared this set to the homeodomain repertoire of species from the other four early branching metazoan lineages. We have identified several important classes and subclasses of homeodomains that appear to be absent from Mnemiopsis and from the poriferan Amphimedon queenslandica. We have also determined that, based on lineage-specific paralog retention and average branch lengths, it is unlikely that these missing classes and subclasses are due to extensive gene loss or unusually high rates of evolution in Mnemiopsis. This paper provides a first glimpse of the first sequenced ctenophore genome. We have characterized the full complement of Mnemiopsis homeodomains from this species and have compared them to species from other early branching lineages. Our results suggest that Porifera and Ctenophora were the first two extant lineages to diverge from the rest of animals. Based on this analysis, we also propose a new name - ParaHoxozoa - for the remaining group that includes Placozoa, Cnidaria and

  7. Microphthalmia in Texel Sheep Is Associated with a Missense Mutation in the Paired-Like Homeodomain 3 (PITX3) Gene

    PubMed Central

    Bürstel, Daniela; Ganter, Martin; Kijas, James; Drögemüller, Cord

    2010-01-01

    Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina's OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G>C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia. PMID:20084168

  8. Microphthalmia in Texel sheep is associated with a missense mutation in the paired-like homeodomain 3 (PITX3) gene.

    PubMed

    Becker, Doreen; Tetens, Jens; Brunner, Adrian; Bürstel, Daniela; Ganter, Martin; Kijas, James; Drögemüller, Cord

    2010-01-13

    Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina's OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G>C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia.

  9. Leaf Rolling Controlled by the Homeodomain Leucine Zipper Class IV Gene Roc5 in Rice1[W

    PubMed Central

    Zou, Liang-ping; Sun, Xue-hui; Zhang, Zhi-guo; Liu, Peng; Wu, Jin-xia; Tian, Cai-juan; Qiu, Jin-long; Lu, Tie-gang

    2011-01-01

    Leaf rolling is considered an important agronomic trait in rice (Oryza sativa) breeding. To understand the molecular mechanism controlling leaf rolling, we screened a rice T-DNA insertion population and isolated the outcurved leaf1 (oul1) mutant showing abaxial leaf rolling. The phenotypes were caused by knockout of Rice outermost cell-specific gene5 (Roc5), an ortholog of the Arabidopsis (Arabidopsis thaliana) homeodomain leucine zipper class IV gene GLABRA2. Interestingly, overexpression of Roc5 led to adaxially rolled leaves, whereas cosuppression of Roc5 resulted in abaxial leaf rolling. Bulliform cell number and size increased in oul1 and Roc5 cosuppression plants but were reduced in Roc5-overexpressing lines. The data indicate that Roc5 negatively regulates bulliform cell fate and development. Gene expression profiling, quantitative polymerase chain reaction, and RNA interference (RNAi) analyses revealed that Protodermal Factor Like (PFL) was probably down-regulated in oul1. The mRNA level of PFL was increased in Roc5-overexpressing lines, and PFL-RNAi transgenic plants exhibit reversely rolling leaves by reason of increases of bulliform cell number and size, indicating that Roc5 may have a conserved function. These are, to our knowledge, the first functional data for a gene encoding a homeodomain leucine zipper class IV transcriptional factor in rice that modulates leaf rolling. PMID:21596949

  10. In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

    PubMed Central

    Fabbro, D; Tell, G; Leonardi, A; Pellizzari, L; Pucillo, C; Lonigro, R; Formisano, S; Damante, G

    1996-01-01

    The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases. PMID:8811078

  11. Identification of candidate downstream genes for the homeodomain transcription factor Labial in Drosophila through oligonucleotide-array transcript imaging

    PubMed Central

    Leemans, Ronny; Loop, Thomas; Egger, Boris; He, Haiqiong; Kammermeier, Lars; Hartmann, Beate; Certa, Ullrich; Reichert, Heinrich; Hirth, Frank

    2001-01-01

    Background: Homeotic genes are key developmental regulators that are highly conserved throughout evolution. Their encoded homeoproteins function as transcription factors to control a wide range of developmental processes. Although much is known about homeodomain-DNA interactions, only a small number of genes acting downstream of homeoproteins have been identified. Here we use a functional genomic approach to identify candidate target genes of the Drosophila homeodomain transcription factor Labial. Results: High-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following labial overexpression in Drosophila embryos. We find significant expression level changes for 96 genes belonging to all functional classes represented on the array. In accordance with our experimental procedure, we expect that these genes are either direct or indirect targets of labial gene action. Among these genes, 48 were upregulated and 48 were downregulated following labial overexpression. This corresponds to 6.3% of the genes represented on the array. For a selection of these genes, we show that the data obtained with the oligonucleotide arrays are consistent with data obtained using quantitative RT-PCR. Conclusions: Our results identify a number of novel candidate downstream target genes for Labial, suggesting that this homeoprotein differentially regulates a limited and distinct set of embryonically expressed Drosophila genes. PMID:11387036

  12. Identification of CtBP1 and CtBP2 as corepressors of zinc finger-homeodomain factor deltaEF1.

    PubMed

    Furusawa, T; Moribe, H; Kondoh, H; Higashi, Y

    1999-12-01

    deltaEF1, a representative of the zinc finger-homeodomain protein family, is a transcriptional repressor which binds E2-box (CACCTG) and related sequences and counteracts the activators through transrepression mechanisms. It has been shown that the N-proximal region of the protein is involved in the transrepression. Here we demonstrate that deltaEF1 has a second mechanism of transrepression recruiting CtBP1 or CtBP2 as its corepressor. A two-hybrid screen of mouse cDNAs with various portions of deltaEF1 identified these proteins, which bind to deltaEF1 in a manner dependent on the PLDLSL sequence located in the short medial (MS) portion of deltaEF1. CtBP1 is the mouse orthologue of human CtBP, known as the C-terminal binding protein of adenovirus E1A, while CtBP2 is the second homologue. Fusion of mouse CtBP1 or CtBP2 to Gal4DBD (Gal4 DNA binding domain) made them Gal4 binding site-dependent transcriptional repressors in transfected 10T1/2 cells, indicating their involvement in a transcriptional repression mechanism. When the MS portion of deltaEF1 was used to Gal4DBD and used to transfect cells, a strong transrepression activity was generated, but this activity was totally dependent on the PLDLSL sequence which served as the site for interaction with endogenous CtBP proteins, indicating that CtBP1 and -2 can act as corepressors. Exogenous CtBP1/2 significantly enhanced transcriptional repression by deltaEF1, and this enhancement was lost if the PLDLSL sequence was altered, demonstrating that CtBP1 and -2 act as corepressors of deltaEF1. In the mouse, CtBP1 is expressed from embryo to adult, but CtBP2 is mainly expressed during embryogenesis. In developing embryos, CtBP1 and CtBP2 are expressed broadly with different tissue preferences. Remarkably, their high expression occurs in subsets of deltaEF1-expressing tissues, e.g., cephalic and dorsal root ganglia, spinal cord, posterior-distal halves of the limb bud mesenchyme, and perichondrium of forming digits

  13. Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development.

    PubMed

    Huang, Mingqian; Sage, Cyrille; Li, Huawei; Xiang, Mengquig; Heller, Stefan; Chen, Zheng-Yi

    2008-11-01

    LIM-homeodomain transcription factors (LIM-HDs) are essential in tissue patterning and differentiation. But their expression patterns in the inner ear are largely unknown. Here we report on a study of twelve LIM-HDs, by their tempo-spatial patterns that imply distinct yet overlapping roles, in the developing mouse inner ear. Expression of Lmx1a and Isl1 begins in the otocyst stage, with Lmx1a exclusively in the non-sensory and Isl1 in the prosensory epithelia. The second wave of expression at E12.5 includes Lhx3, 5, 9, Isl2, and Lmx1b in the differentiating sensory epithelia with cellular specificities. With the exception of Lmx1a and Lhx3, all LIM-HDs are expressed in ganglion neurons. Expression of multiple LIM-HDs within a cell type suggests their redundant function.

  14. Existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot

    PubMed Central

    Kheirollahi, Majid; Khosravi, Fereshteh; Ashouri, Saeideh; Ahmadi, Alireza

    2016-01-01

    Background: Tetralogy of Fallot (TOF), the most common cyanotic heart defect and one of the most common congenital heart diseases, occurs mostly sporadically and nonsyndromically. The underlying molecular genetic mechanism is not known. Therefore, the existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot is evaluated. Materials and Methods: In the present study, we analyzed the peripheral blood samples of27 patients in order to find any mutation in the 180 bp homeodomain-encoding region of NKX2.5 gene, which is known to be involved in heart development and diseases. DNA was extracted and all the samples were amplified by polymerase chain reaction (PCR) and sequenced. Results: Twenty-seven patients were included in the study. Twenty-five of them were infants and children (6 days to 11 years of age), one was a teenager (14-years of age), and another was a 33-year-old man [mean ± standard deviation (SD): 5.80 ± 3.90 years]. Thirteen patents were males (mean ± SD: 6.587077 ± 5.02 years) and 14 were females (mean ± SD: 5.0726 ± 2.81 years). One synonymous variant, i.e., c.543G>A was identified in one patient. Conclusion: Mutations in the homeodomain-encoding region of NKX2.5 gene may not have an outstanding role in etiology of tetralogy of Fallot patients in Iran. PMID:27904570

  15. Homeodomain protein Otp affects developmental neuropeptide switching in oxytocin neurons associated with a long-term effect on social behavior

    PubMed Central

    Wircer, Einav; Blechman, Janna; Borodovsky, Nataliya; Tsoory, Michael; Nunes, Ana Rita; Oliveira, Rui F; Levkowitz, Gil

    2017-01-01

    Proper response to stress and social stimuli depends on orchestrated development of hypothalamic neuronal circuits. Here we address the effects of the developmental transcription factor orthopedia (Otp) on hypothalamic development and function. We show that developmental mutations in the zebrafish paralogous gene otpa but not otpb affect both stress response and social preference. These behavioral phenotypes were associated with developmental alterations in oxytocinergic (OXT) neurons. Thus, otpa and otpb differentially regulate neuropeptide switching in a newly identified subset of OXT neurons that co-express the corticotropin-releasing hormone (CRH). Single-cell analysis revealed that these neurons project mostly to the hindbrain and spinal cord. Ablation of this neuronal subset specifically reduced adult social preference without affecting stress behavior, thereby uncoupling the contribution of a specific OXT cluster to social behavior from the general otpa−/− deficits. Our findings reveal a new role for Otp in controlling developmental neuropeptide balance in a discrete OXT circuit whose disrupted development affects social behavior. DOI: http://dx.doi.org/10.7554/eLife.22170.001 PMID:28094761

  16. The homeodomain protein CePHOX2/CEH-17 controls antero-posterior axonal growth in C. elegans.

    PubMed

    Pujol, N; Torregrossa, P; Ewbank, J J; Brunet, J F

    2000-08-01

    An essential aspect of a neuron's identity is the pattern of its axonal projections. In C. elegans, axons extend either longitudinally or circumferentially in response to distinct molecular cues, some of which have been identified. It is currently unclear, however, how the differential capacity to respond to these cues is transcriptionally implemented in distinct neuronal subtypes. Here, we characterise a C. elegans paired-like homeobox gene, CePhox2/ceh-17, expressed in five head neurons, ALA and the 4 SIAs, all of which project axons towards the tail along the lateral and sublateral cords. Abrogation of ceh-17 function, while leaving intact many phenotypic traits of these neurons, disrupts their antero-posterior axonal elongation beyond the mid-body region. Conversely, ectopic expression of ceh-17 in the mechanoreceptors, several of which are known to pioneer their tract, leads to exaggerated longitudinal axonal outgrowth. Thus, ceh-17 is a novel gene involved in fasciculation-independent longitudinal axonal navigation.

  17. Rice Homeodomain Protein WOX11 Recruits a Histone Acetyltransferase Complex to Establish Programs of Cell Proliferation of Crown Root Meristem.

    PubMed

    Zhou, Shaoli; Jiang, Wei; Long, Fei; Cheng, Saifeng; Yang, Wenjing; Zhao, Yu; Zhou, Dao-Xiu

    2017-05-01

    Shoot-borne crown roots are the major root system in cereals. Previous work has shown that the Wuschel-related homeobox gene WOX11 is necessary and sufficient to promote rice (Oryza sativa) crown root emergence and elongation. Here, we show that WOX11 recruits the ADA2-GCN5 histone acetyltransferase module to activate downstream target genes in crown root meristem. Rice ADA2 and GCN5 genes are highly expressed in root meristem and are shown to be essential for cell division and growth. WOX11 and ADA2-GCN5 commonly target and regulate a set of root-specific genes involved in energy metabolism, cell wall biosynthesis, and hormone response, some of which are known to be important for root development. The results indicate that the recruitment of ADA2-GCN5 by WOX11 establishes gene expression programs of crown root meristem cell division and suggest that permissive chromatin modification involving histone acetylation is a strategy for WOX11 to stimulate root meristem development. © 2017 American Society of Plant Biologists. All rights reserved.

  18. A petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence

    USDA-ARS?s Scientific Manuscript database

    Flower senescence is mediated in part by changes of plant hormones, such as ethylene, cytokinin and abscisic acid (ABA). Ethylene is known to control flower senescence in many species, especially ethylene sensitive flowers, like petunia, carnation and rose. During flower senescence in petunia and ot...

  19. Auxin Response Factor2 (ARF2) and Its Regulated Homeodomain Gene HB33 Mediate Abscisic Acid Response in Arabidopsis

    PubMed Central

    Wang, Li; Hua, Deping; He, Junna; Duan, Ying; Chen, Zhizhong; Hong, Xuhui; Gong, Zhizhong

    2011-01-01

    The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth. PMID:21779177

  20. The homeodomain transcription factor Hb9 controls axon guidance in Drosophila through the regulation of Robo receptors.

    PubMed

    Santiago, Celine; Labrador, Juan-Pablo; Bashaw, Greg J

    2014-04-10

    Transcription factors establish neural diversity and wiring specificity; however, how they orchestrate changes in cell morphology remains poorly understood. The Drosophila Roundabout (Robo) receptors regulate connectivity in the CNS, but how their precise expression domains are established is unknown. Here, we show that the homeodomain transcription factor Hb9 acts upstream of Robo2 and Robo3 to regulate axon guidance in the Drosophila embryo. In ventrally projecting motor neurons, hb9 is required for robo2 expression, and restoring Robo2 activity in hb9 mutants rescues motor axon defects. Hb9 requires its conserved repressor domain and functions in parallel with Nkx6 to regulate robo2. Moreover, hb9 can regulate the medio-lateral position of axons through robo2 and robo3, and restoring robo3 expression in hb9 mutants rescues the lateral position defects of a subset of neurons. Altogether, these data identify Robo2 and Robo3 as key effectors of Hb9 in regulating nervous system development.

  1. Transcriptional dominance of Pax7 in adult myogenesis is due to high-affinity recognition of homeodomain motifs.

    PubMed

    Soleimani, Vahab D; Punch, Vincent G; Kawabe, Yoh-ichi; Jones, Andrew E; Palidwor, Gareth A; Porter, Christopher J; Cross, Joe W; Carvajal, Jaime J; Kockx, Christel E M; van IJcken, Wilfred F J; Perkins, Theodore J; Rigby, Peter W J; Grosveld, Frank; Rudnicki, Michael A

    2012-06-12

    Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrate distinct attributes of Pax7 function and provide mechanistic insight into the nonredundancy of Pax3 and Pax7 in muscle development. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Micro-trichome as a class I homeodomain-leucine zipper gene regulates multicellular trichome development in Cucumis sativus.

    PubMed

    Zhao, Jun-Long; Pan, Jun-Song; Guan, Yuan; Zhang, Wei-Wei; Bie, Bei-Bei; Wang, Yun-Li; He, Huan-Le; Lian, Hong-Li; Cai, Run

    2015-11-01

    Plant trichomes serve as a highly suitable model for investigating cell differentiation at the single-cell level. The regulatory genes involved in unicellular trichome development in Arabidopsis thaliana have been intensively studied, but genes regulating multicellular trichome development in plants remain unclear. Here, we characterized Cucumis sativus (cucumber) trichomes as representative multicellular and unbranched structures, and identified Micro-trichome (Mict), using map-based cloning in an F2 segregating population of 7,936 individuals generated from a spontaneous mict mutant. In mict plants, trichomes in both leaves and fruits, are small, poorly developed, and denser than in the wild type. Sequence analysis revealed that a 2,649-bp genomic deletion, spanning the first and second exons, occurred in a plant-specific class I homeodomain-leucine zipper gene. Tissue-specific expression analysis indicated that Mict is strongly expressed in the trichome cells. Transcriptome profiling identified potential targets of Mict including putative homologs of genes known in other systems to regulate trichome development, meristem determinacy, and hormone responsiveness. Phylogenic analysis charted the relationships among putative homologs in angiosperms. Our paper represents initial steps toward understanding the development of multicellular trichomes.

  3. Organisation of the lamprey (Lampetra fluviatilis) embryonic brain: insights from LIM-homeodomain, Pax and hedgehog genes.

    PubMed

    Osorio, Joana; Mazan, Sylvie; Rétaux, Sylvie

    2005-12-01

    To investigate the embryonic development of the central nervous system of the lamprey Lampetra fluviatilis, we have isolated and analysed the expression patterns of members of the LIM-homeodomain, Pax, Hedgehog and Nkx2.1 families. Using degenerate RT-PCR, single representatives of Lhx1/Lhx5, Lhx2/Lhx9, Pax3/Pax7 and Hedgehog families could be isolated in L. fluviatilis. Expression analysis revealed that the lamprey forebrain presents a clear neuromeric pattern. We describe the existence of 4 embryonic diencephalic prosomeres whose boundaries can be identified by the combined and relative expressions of LfPax37, LfLhx15 and LfLhx29. This suggests that the embryonic lamprey and gnathostome forebrain are patterned in a highly similar manner. Moreover, analysis of the LfHh gene, which is expressed in the hypothalamus, zona limitans intrathalamica and floor plate, reveals the possible molecular origin of this neuromeric brain pattern. By contrast, LfHh and LfNkx2.1 expressions suggest major differences in patterning mechanisms of the ventral telencephalon when compared to gnathostomes. In summary, our findings highlight a neuromeric organisation of the embryonic agnathan forebrain and point to the possible origin of this organisation, which is thus a truly vertebrate character. They also suggest that Hh/Shh midline signalling might act as a driving force for forebrain evolution.

  4. Complex organizational defects of fibroblast architecture in the mouse spleen with Nkx2.3 homeodomain deficiency.

    PubMed

    Bovári, Judit; Czömpöly, Tamás; Olasz, Katinka; Arnold, Hans-Henning; Balogh, Péter

    2007-01-01

    The capacity of secondary lymphoid organs to provide suitable tissue environment for mounting immune responses is dependent on their compartmentalized stromal constituents, including distinct fibroblasts. In addition to various members of the tumor necrosis factor/lymphotoxin beta family as important morphogenic regulators of peripheral lymphoid tissue development, the formation of stromal elements of spleen is also influenced by the Nkx2.3 homeodomain transcription factor in a tissue-specific fashion. Here we extend our previous work on the role of Nkx2.3-mediated regulation in the development of spleen architecture by analyzing the structure of reticular fibroblastic meshwork of spleen in inbred Nkx2.3-deficient mice. Using immunohistochemistry and dual-label immunofluorescence we found both distributional abnormalities, manifested as poor reticular compartmentalization of T-zone and circumferential reticulum, and developmental blockade, resulting in the absence of a complementary fibroblast subpopulation of white pulp. The disregulated distribution of fibroblasts was accompanied with an increased binding of immunohistochemically detectable complement factor C4 by T-cell zone-associated reticular fibroblasts, distinct from follicular dendritic cells with inherently high-level expression of bound C4. These data indicate that the impact of Nkx2.3 gene deficiency on fibroblast ontogeny within the spleen extends beyond its distributional effects, and that the formation of various white pulp fibroblast subsets is differentially affected by the presence of Nkx2.3 activity, possibly also influencing their role in various immune functions linked with complement activation and deposition.

  5. Genome-Wide Identification, Evolution and Expression Analysis of the Grape (Vitis vinifera L.) Zinc Finger-Homeodomain Gene Family

    PubMed Central

    Wang, Hao; Yin, Xiangjing; Li, Xiaoqin; Wang, Li; Zheng, Yi; Xu, Xiaozhao; Zhang, Yucheng; Wang, Xiping

    2014-01-01

    Plant zinc finger-homeodomain (ZHD) genes encode a family of transcription factors that have been demonstrated to play an important role in the regulation of plant growth and development. In this study, we identified a total of 13 ZHD genes (VvZHD) in the grape genome that were further classified into at least seven groups. Genome synteny analysis revealed that a number of VvZHD genes were present in the corresponding syntenic blocks of Arabidopsis, indicating that they arose before the divergence of these two species. Gene expression analysis showed that the identified VvZHD genes displayed distinct spatiotemporal expression patterns, and were differentially regulated under various stress conditions and hormone treatments, suggesting that the grape VvZHDs might be also involved in plant response to a variety of biotic and abiotic insults. Our work provides insightful information and knowledge about the ZHD genes in grape, which provides a framework for further characterization of their roles in regulation of stress tolerance as well as other aspects of grape productivity. PMID:24705465

  6. Homeodomain-containing gene 10 inhibits cell apoptosis and promotes cell invasion and migration in osteosarcoma cell lines.

    PubMed

    Xiong, Wen; Zhou, Quan; Liu, Gang; Liu, Xiang-Sheng; Li, Xin-Yu

    2017-05-01

    Homeodomain-containing gene 10 (HOXC10) belongs to the homeobox family, which encodes a highly conserved family of transcription factors that plays an important role in morphogenesis in all multicellular organisms. Altered expressions of HOXC10 have been reported in several malignancies. This study was aimed to reveal the expression profile of HOXC10 in osteosarcoma and evaluated whether HOXC10 is a molecular target for cancer therapy. We found that HOXC10 was up-regulated in osteosarcoma tissues compared with bone cyst specimens from The Cancer Genome Atlas database. Osteosarcoma MG63 cells were infected with HOXC10 shRNA expressing vector, and 143B cells were infected with HOXC10 expressing vector. We found that reduced expression of HOXC10 markedly impaired the ability of proliferation, invasion, and migration, and promoted cell apoptosis in vitro and in vivo. Up-regulated expression of HOXC10 promoted the proliferation, invasion, and migration, and inhibited apoptosis of 143B cells. Additionally, HOXC10 regulated apoptosis and migration via modulating expression of Bax/Bcl-2, caspase-3, MMP-2/MMP-9, and E-cadherin in both MG63 and 143B cells and in vivo. These results indicated that HOXC10 might be a diagnostic marker for osteosarcoma and could be a potential molecular target for the therapy of osteosarcoma.

  7. Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.

    PubMed

    Szeto, D P; Rodriguez-Esteban, C; Ryan, A K; O'Connell, S M; Liu, F; Kioussi, C; Gleiberman, A S; Izpisúa-Belmonte, J C; Rosenfeld, M G

    1999-02-15

    Pitx1 is a Bicoid-related homeodomain factor that exhibits preferential expression in the hindlimb, as well as expression in the developing anterior pituitary gland and first branchial arch. Here, we report that Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of the hindlimb, resulting in a limb that exhibits structural changes in tibia and fibula as well as patterning alterations in patella and proximal tarsus, to more closely resemble the corresponding forelimb structures. Deletion of the Pitx1 locus results in decreased distal expression of the hindlimb-specific marker, the T-box factor, Tbx4. On the basis of similar expression patterns in chick, targeted misexpression of chick Pitx1 in the developing wing bud causes the resulting limb to assume altered digit number and morphogenesis, with Tbx4 induction. We hypothesize that Pitx1 serves to critically modulate morphogenesis, growth, and potential patterning of a specific hindlimb region, serving as a component of the morphological and growth distinctions in forelimb and hindlimb identity. Pitx1 gene-deleted mice also exhibit reciprocal abnormalities of two ventral and one dorsal anterior pituitary cell types, presumably on the basis of its synergistic functions with other transcription factors, and defects in the derivatives of the first branchial arch, including cleft palate, suggesting a proliferative defect in these organs analogous to that observed in the hindlimb.

  8. Genome-wide identification, evolution and expression analysis of the grape (Vitis vinifera L.) zinc finger-homeodomain gene family.

    PubMed

    Wang, Hao; Yin, Xiangjing; Li, Xiaoqin; Wang, Li; Zheng, Yi; Xu, Xiaozhao; Zhang, Yucheng; Wang, Xiping

    2014-04-03

    Plant zinc finger-homeodomain (ZHD) genes encode a family of transcription factors that have been demonstrated to play an important role in the regulation of plant growth and development. In this study, we identified a total of 13 ZHD genes (VvZHD) in the grape genome that were further classified into at least seven groups. Genome synteny analysis revealed that a number of VvZHD genes were present in the corresponding syntenic blocks of Arabidopsis, indicating that they arose before the divergence of these two species. Gene expression analysis showed that the identified VvZHD genes displayed distinct spatiotemporal expression patterns, and were differentially regulated under various stress conditions and hormone treatments, suggesting that the grape VvZHDs might be also involved in plant response to a variety of biotic and abiotic insults. Our work provides insightful information and knowledge about the ZHD genes in grape, which provides a framework for further characterization of their roles in regulation of stress tolerance as well as other aspects of grape productivity.

  9. The PAX3 paired domain and homeodomain function as a single binding module in vivo to regulate subnuclear localization and mobility by a mechanism that requires base-specific recognition.

    PubMed

    Corry, Gareth N; Raghuram, Nikhil; Missiaen, Kristal K; Hu, Ninghe; Hendzel, Michael J; Underhill, D Alan

    2010-09-10

    The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.

  10. The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes

    SciTech Connect

    Rascle, Anne Neumann, Tanja; Raschta, Anne-Sarah; Neumann, Astrid; Heining, Eva; Kastner, Juergen; Witzgall, Ralph

    2009-01-01

    LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-{kappa}B target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-{kappa}B activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-{kappa}B activity by TNF-{alpha} synergizes induction of NF-{kappa}B target genes by LMX1B. Chromatin immunoprecipitation demonstrated that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized {kappa}B site, and that LMX1B recruitment correlates with increased NF-{kappa}B DNA association. IL-6 promoter-reporter assays showed that the {kappa}B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-{kappa}B target genes is affected in the kidney of Lmx1b{sup -/-} knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-{kappa}B target genes in cooperation with nuclear p50/p65 NF-{kappa}B.

  11. Multifaceted Histone H3 Methylation and Phosphorylation Readout by the Plant Homeodomain Finger of Human Nuclear Antigen Sp100C.

    PubMed

    Zhang, Xiaojie; Zhao, Dan; Xiong, Xiaozhe; He, Zhimin; Li, Haitao

    2016-06-10

    The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation.

  12. Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm.

    PubMed

    Frazee, Richard W; Taylor, Jennifer A; Tullius, Thomas D

    2002-11-01

    The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm

  13. Distinct Behaviour of the Homeodomain Derived Cell Penetrating Peptide Penetratin in Interaction with Different Phospholipids

    PubMed Central

    Maniti, Ofelia; Alves, Isabel; Trugnan, Germain; Ayala-Sanmartin, Jesus

    2010-01-01

    Background Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial. Methodology/Principal Findings In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG) to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β–sheet contributions. Conclusions/Significance These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import) and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution. PMID:21209890

  14. The identification of Cucumis sativus Glabrous 1 (CsGL1) required for the formation of trichomes uncovers a novel function for the homeodomain-leucine zipper I gene.

    PubMed

    Li, Qiang; Cao, Chenxing; Zhang, Cunjia; Zheng, Shuangshuang; Wang, Zenghui; Wang, Lina; Ren, Zhonghai

    2015-05-01

    The spines and bloom of cucumber (Cucumis sativus L.) fruit are two important quality traits related to fruit market value. However, until now, none of the genes involved in the formation of cucumber fruit spines and bloom trichomes has been identified. Here, the characterization of trichome development in wild-type (WT) cucumber and a spontaneous mutant, glabrous 1 (csgl1) controlled by a single recessive nuclear gene, with glabrous aerial organs, is reported. Via map-based cloning, CsGL1 was isolated and it was found that it encoded a member of the homeodomain-leucine zipper I (HD-Zip I) proteins previously identified to function mainly in the abiotic stress responses of plants. Tissue-specific expression analysis indicated that CsGL1 was strongly expressed in trichomes and fruit spines. In addition, CsGL1 was a nuclear protein with weak transcriptional activation activity in yeast. A comparative analysis of the digital gene expression (DGE) profile between csgl1 and WT leaves revealed that CsGL1 had a significant influence on the gene expression profile in cucumber, especially on genes related to cellular process, which is consistent with the phenotypic difference between csgl1 and the WT. Moreover, two genes, CsMYB6 and CsGA20ox1, possibly involved in the formation of cucumber trichomes and fruit spines, were characterized. Overall, the findings reveal a new function for the HD-Zip I gene subfamily, and provide some candidate genes for genetic engineering approaches to improve cucumber fruit external quality.

  15. LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori.

    PubMed

    Kimoto, Mai; Tsubota, Takuya; Uchino, Keiro; Sezutsu, Hideki; Takiya, Shigeharu

    2015-01-01

    In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Methods for molecular dynamics simulations of protein folding/unfolding in solution.

    PubMed

    Beck, David A C; Daggett, Valerie

    2004-09-01

    All atom molecular dynamics simulations have become a standard method for mapping equilibrium protein dynamics and non-equilibrium events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and analysis derived from previous studies are also presented. As a measure of validation, our water model is compared with experiment. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the experimental evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by experiment occur on timescales accessible with the high-resolution molecular dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temperature quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described.

  17. PAX proteins and fables of their reconstruction.

    PubMed

    Underhill, Darrell Alan

    2012-01-01

    The PAX proteins derive their name from the "paired box," a region of homology first described between the Drosophila paired (Prd) and gooseberry (Gsb) proteins and later found to encode a sequence-specific DNA-binding activity. Both Prd and Gsb also contain a homeodomain, and this combination of DNA-binding domains is conserved in ancestral predecessors, reflecting an early "homeodomain-capturing" event. In addition, the prototypic PAX protein was thought to contain 2 additional features, namely the octapeptide (or eh1) motif and PHT (or OAR) domain-both modulate PAX regulatory activity but are not unique to the PAX family. Together with gene duplications and mutagenesis, a domain loss model accounts for the distinct architecture and sequence of extant PAX proteins. Despite the disparate evolutionary history of these 4 conserved motifs, there is a remarkable level of interplay that is modulated by discrete sequences elsewhere in the protein. Here, the implications with respect to the evolution of PAX protein structure and activity are discussed, it is suggested that the sum of these constituent domains is more than the contribution of individual parts. When combined with alternative splicing and posttranslational modifications, this model confers an extraordinary degree of functional diversity to even highly related PAX proteins.

  18. Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein.

    PubMed

    Showalter, Aaron D; Smith, Timothy P L; Bennett, Gary L; Sloop, Kyle W; Whitsett, Julie A; Rhodes, Simon J

    2002-05-29

    The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.

  19. Overexpression of the Epidermis-Specific Homeodomain-Leucine Zipper IV Transcription Factor OUTER CELL LAYER1 in Maize Identifies Target Genes Involved in Lipid Metabolism and Cuticle Biosynthesis1[C][W

    PubMed Central

    Javelle, Marie; Vernoud, Vanessa; Depège-Fargeix, Nathalie; Arnould, Christine; Oursel, Delphine; Domergue, Frédéric; Sarda, Xavier; Rogowsky, Peter M.

    2010-01-01

    Transcription factors of the homeodomain-leucine zipper IV (HD-ZIP IV) family play crucial roles in epidermis-related processes. To gain further insight into the molecular function of OUTER CELL LAYER1 (OCL1), 14 target genes up- or down-regulated in transgenic maize (Zea mays) plants overexpressing OCL1 were identified. The 14 genes all showed partial coexpression with OCL1 in maize organs, and several of them shared preferential expression in the epidermis with OCL1. They encoded proteins involved in lipid metabolism, defense, envelope-related functions, or cuticle biosynthesis and include ZmWBC11a (for white brown complex 11a), an ortholog of AtWBC11 involved in the transport of wax and cutin molecules. In support of the annotations, OCL1-overexpressing plants showed quantitative and qualitative changes of cuticular wax compounds in comparison with wild-type plants. An increase in C24 to C28 alcohols was correlated with the transcriptional up-regulation of ZmFAR1, coding for a fatty acyl-coenzyme A reductase. Transcriptional activation of ZmWBC11a by OCL1 was likely direct, since transactivation in transiently transformed maize kernels was abolished by a deletion of the activation domain in OCL1 or mutations in the L1 box, a cis-element bound by HD-ZIP IV transcription factors. Our data demonstrate that, in addition to AP2/EREBP and MYB-type transcription factors, members of the HD-ZIP IV family contribute to the transcriptional regulation of genes involved in cuticle biosynthesis. PMID:20605912

  20. Overexpression of the epidermis-specific homeodomain-leucine zipper IV transcription factor Outer Cell Layer1 in maize identifies target genes involved in lipid metabolism and cuticle biosynthesis.

    PubMed

    Javelle, Marie; Vernoud, Vanessa; Depège-Fargeix, Nathalie; Arnould, Christine; Oursel, Delphine; Domergue, Frédéric; Sarda, Xavier; Rogowsky, Peter M

    2010-09-01

    Transcription factors of the homeodomain-leucine zipper IV (HD-ZIP IV) family play crucial roles in epidermis-related processes. To gain further insight into the molecular function of OUTER CELL LAYER1 (OCL1), 14 target genes up- or down-regulated in transgenic maize (Zea mays) plants overexpressing OCL1 were identified. The 14 genes all showed partial coexpression with OCL1 in maize organs, and several of them shared preferential expression in the epidermis with OCL1. They encoded proteins involved in lipid metabolism, defense, envelope-related functions, or cuticle biosynthesis and include ZmWBC11a (for white brown complex 11a), an ortholog of AtWBC11 involved in the transport of wax and cutin molecules. In support of the annotations, OCL1-overexpressing plants showed quantitative and qualitative changes of cuticular wax compounds in comparison with wild-type plants. An increase in C24 to C28 alcohols was correlated with the transcriptional up-regulation of ZmFAR1, coding for a fatty acyl-coenzyme A reductase. Transcriptional activation of ZmWBC11a by OCL1 was likely direct, since transactivation in transiently transformed maize kernels was abolished by a deletion of the activation domain in OCL1 or mutations in the L1 box, a cis-element bound by HD-ZIP IV transcription factors. Our data demonstrate that, in addition to AP2/EREBP and MYB-type transcription factors, members of the HD-ZIP IV family contribute to the transcriptional regulation of genes involved in cuticle biosynthesis.

  1. PBX and MEIS as Non-DNA-Binding Partners in Trimeric Complexes with HOX Proteins

    PubMed Central

    Shanmugam, Kandavel; Green, Nancy C.; Rambaldi, Isabel; Saragovi, H. Uri; Featherstone, Mark S.

    1999-01-01

    HOX, PBX, and MEIS transcription factors bind DNA through a homeodomain. PBX proteins bind DNA cooperatively as heterodimers with MEIS family members and also with HOX proteins from paralog groups 1 to 10. MEIS proteins cooperatively bind DNA with ABD-B class HOX proteins of groups 9 and 10. Here, we examine aspects of dimeric and higher-order interactions between these three homeodomain classes. The most significant results can be summarized as follows. (i) Most of PBX N terminal to the homeodomain is required for efficient cooperative binding with HOXD4 and HOXD9. (ii) MEIS and PBX proteins form higher-order complexes on a heterodimeric binding site. (iii) Although MEIS does not cooperatively bind DNA with ANTP class HOX proteins, it does form a trimer as a non-DNA-binding partner with DNA-bound PBX-HOXD4. (iv) The N terminus of HOXD4 negatively regulates trimer formation. (v) MEIS forms a similar trimer with DNA-bound PBX-HOXD9. (vi) A related trimer (where MEIS is a non-DNA-binding partner) is formed on a transcriptional promoter within the cell. (vii) We observe an additional trimer class involving non-DNA-bound PBX and DNA-bound MEIS-HOXD9 or MEIS-HOXD10 heterodimers that is enhanced by mutation of the PBX homeodomain. (viii) In this latter trimer, PBX is likely to contact both MEIS and HOXD9/D10. (ix) The stability of DNA binding by all trimers is enhanced relative to the heterodimers. These findings suggest novel functions for PBX and MEIS in modulating the function of DNA-bound MEIS-HOX and PBX-HOX heterodimers, respectively. PMID:10523646

  2. PBX and MEIS as non-DNA-binding partners in trimeric complexes with HOX proteins.

    PubMed

    Shanmugam, K; Green, N C; Rambaldi, I; Saragovi, H U; Featherstone, M S

    1999-11-01

    HOX, PBX, and MEIS transcription factors bind DNA through a homeodomain. PBX proteins bind DNA cooperatively as heterodimers with MEIS family members and also with HOX proteins from paralog groups 1 to 10. MEIS proteins cooperatively bind DNA with ABD-B class HOX proteins of groups 9 and 10. Here, we examine aspects of dimeric and higher-order interactions between these three homeodomain classes. The most significant results can be summarized as follows. (i) Most of PBX N terminal to the homeodomain is required for efficient cooperative binding with HOXD4 and HOXD9. (ii) MEIS and PBX proteins form higher-order complexes on a heterodimeric binding site. (iii) Although MEIS does not cooperatively bind DNA with ANTP class HOX proteins, it does form a trimer as a non-DNA-binding partner with DNA-bound PBX-HOXD4. (iv) The N terminus of HOXD4 negatively regulates trimer formation. (v) MEIS forms a similar trimer with DNA-bound PBX-HOXD9. (vi) A related trimer (where MEIS is a non-DNA-binding partner) is formed on a transcriptional promoter within the cell. (vii) We observe an additional trimer class involving non-DNA-bound PBX and DNA-bound MEIS-HOXD9 or MEIS-HOXD10 heterodimers that is enhanced by mutation of the PBX homeodomain. (viii) In this latter trimer, PBX is likely to contact both MEIS and HOXD9/D10. (ix) The stability of DNA binding by all trimers is enhanced relative to the heterodimers. These findings suggest novel functions for PBX and MEIS in modulating the function of DNA-bound MEIS-HOX and PBX-HOX heterodimers, respectively.

  3. Genome-wide DNA binding pattern of the homeodomain transcription factor Sine oculis (So) in the developing eye of Drosophila melanogaster.

    PubMed

    Jusiak, Barbara; Wang, Feng; Karandikar, Umesh C; Kwak, Su-Jin; Wang, Hui; Chen, Rui; Mardon, Graeme

    2014-12-01

    The eye of the fruit fly Drosophila melanogaster provides a highly tractable genetic model system for the study of animal development, and many genes that regulate Drosophila eye formation have homologs implicated in human development and disease. Among these is the homeobox gene sine oculis (so), which encodes a homeodomain transcription factor (TF) that is both necessary for eye development and sufficient to reprogram a subset of cells outside the normal eye field toward an eye fate. We have performed a genome-wide analysis of So binding to DNA prepared from developing Drosophila eye tissue in order to identify candidate direct targets of So-mediated transcriptional regulation, as described in our recent article [1]. The data are available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE52943. Here we describe the methods, data analysis, and quality control of our So ChIP-seq dataset.

  4. Gsh-4 encodes a LIM-type homeodomain, is expressed in the developing central nervous system and is required for early postnatal survival.

    PubMed Central

    Li, H; Witte, D P; Branford, W W; Aronow, B J; Weinstein, M; Kaur, S; Wert, S; Singh, G; Schreiner, C M; Whitsett, J A

    1994-01-01

    We present an initial characterization of the murine Gsh-4 gene which is shown to encode a LIM-type homeodomain. Genes in this category are known to control late developmental cell-type specification events in simpler organisms. Whole mount and serial section in situ hybridizations show transient Gsh-4 expression in ventrolateral regions of the developing neural tube and hindbrain. Mice homozygous for a targeted mutation in Gsh-4 suffer early postnatal death resulting from immature lungs which do not inflate. Prenatal administration of progesterone and glucocorticoid, to extend gestational term and accelerate maturation, resulted in lung inflation at birth. Nevertheless, the hormonally treated mutants generally failed to survive beyond an hour after birth, due to ineffective breathing efforts. It is concluded that Gsh-4 plays a critical role in the development of respiratory control mechanisms and in the normal growth and maturation of the lung. Images PMID:7913017

  5. The WD-Repeat Protein CsTTG1 Regulates Fruit Wart Formation through Interaction with the Homeodomain-Leucine Zipper I Protein Mict1

    PubMed Central

    Yin, Shuai; Liu, Xingwang; Liu, Bin; Yang, Sen; Xue, Shudan; Cai, Yanling; Liu, Huiling; Dong, Mingming; Zhang, Yaqi; Zhao, Binyu

    2016-01-01

    The cucumber (Cucumis sativus) fruit is covered with bloom trichomes and warts (composed of spines and tubercules), which have an important impact on the commercial value of the crop. However, little is known about the regulatory mechanism underlying their formation. Here, we reported that the cucumber WD-repeat homolog CsTTG1, which is localized in the nucleus and cytomembrane, plays an important role in the formation of cucumber fruit bloom trichomes and warts. Functional characterization of CsTTG1 revealed that it is mainly expressed in the epidermis of cucumber ovary and that its overexpression in cucumber alters the density of fruit bloom trichomes and spines, thereby promoting the warty fruit trait. Conversely, silencing CsTTG1 expression inhibits the initiation of fruit spines. Molecular and genetic analyses showed that CsTTG1 acts in parallel to Mict/CsGL1, a key trichome formation factor, to regulate the initiation of fruit trichomes, including fruit bloom trichomes and spines, and that the further differentiation of fruit spines and formation of tubercules regulated by CsTTG1 is dependent on Mict. Using yeast two-hybrid assay and bimolecular fluorescence complementation assay, we determined that CsTTG1 directly interacts with Mict. Collectively, our results indicate that CsTTG1 is an important component of the molecular network that regulates fruit bloom trichome and wart formation in cucumber. PMID:27208299

  6. Mouse Model of Human Congenital Heart Disease: Progressive Atrioventricular Block Induced by a Heterozygous Nkx2-5 Homeodomain Missense Mutation.

    PubMed

    Chowdhury, Rajib; Ashraf, Hassan; Melanson, Michelle; Tanada, Yohei; Nguyen, Minh; Silberbach, Michael; Wakimoto, Hiroko; Benson, D Woodrow; Anderson, Robert H; Kasahara, Hideko

    2015-10-01

    Heterozygous human NKX2-5 homeodomain (DNA-binding domain) missense mutations are highly penetrant for varied congenital heart defects, including progressive atrioventricular (AV) block requiring pacemaker implantation. We recently replicated this genetic defect in a murine knockin model, in which we demonstrated highly penetrant, pleiotropic cardiac anomalies. In this study, we examined postnatal AV conduction in the knockin mice. A murine knockin model (Arg52Gly, Nkx2-5(+/R52G)) in a 129/Sv background was analyzed by histopathology, surface, and telemetry ECG, and in vivo electrophysiology studies, comparing with control Nkx2-5(+/+) mice at diverse postnatal stages, ranging from postnatal day 1 (P1) to 17 months. PR prolongation (first degree AV block) was present at 4 weeks, 7 months, and 17 months of age, but not at P1 in the mutant mice. Advanced AV block was also occasionally demonstrated in the mutant mice. Electrophysiology studies showed that AV nodal function and right ventricular effective refractory period were impaired in the mutant mice, whereas sinus nodal function was not affected. AV nodal size was significantly smaller in the mutant mice than their controls at 4 weeks of age, corresponding to the presence of PR prolongation, but not P1, suggesting, at least in part, that the conduction abnormalities are the result of a morphologically atrophic AV node. The highly penetrant and progressive AV block phenotype seen in human heterozygous missense mutations in NKX2-5 homeodomain was replicated in mice by knocking in a comparable missense mutation. © 2015 American Heart Association, Inc.

  7. Combined pituitary hormone deficiency caused by a novel mutation of a highly conserved residue (F88S) in the homeodomain of PROP-1.

    PubMed

    Osorio, M G; Kopp, P; Marui, S; Latronico, A C; Mendonca, B B; Arnhold, I J

    2000-08-01

    Mutations in the pituitary-specific paired-like homeodomain transcription factor, PROP-1, result in combined pituitary hormone deficiency. We studied a Brazilian girl, offspring of first cousins, who presented with short stature and deficiencies of GH, TSH, PRL, LH, and FSH. Her cortisol response to hypoglycemia was determined at age 4.9, 10.7, and 14.1 yr and remained normal. Magnetic resonance imaging at the age of 9 yr revealed an anterior pituitary lobe of diminished height (3 mm; normal, 4.5 +/- 0.6), but radiography revealed a sella turcica volume above the normal mean. Direct sequencing of the PROP-1 gene revealed homozygosity for a novel 263T>C transition that results in the replacement of a highly conserved phenylalanine by serine at codon 88 (F88S). F88 constitutes the hydrophobic core of the first helix of the homeodomain of PROP-1, and the substitution by the polar residue serine is expected to alter the secondary structure and impair binding of the mutated PROP-1 to DNA target sequences. The F88S mutation (which corresponds to murine F85S) was introduced into the murine Prop-1 complementary DNA and its consequences on DNA binding and trans-activation were assessed in vitro. In contrast to wild-type Prop-1, the F88S mutant showed no significant DNA binding to a PRDQ9 Prop-1 response element in gel shift assays. Transcriptional activation of a luciferase reporter gene containing a PRDQ9 site upstream of a simian virus 40 promoter was reduced to approximately 34% compared with that of wild-type Prop-1 in transiently transfected TSA-201 human embryonic kidney cells. The F88S mutation further expands the repertoire of mutations in PROP-1.

  8. PBX proteins: much more than Hox cofactors.

    PubMed

    Laurent, Audrey; Bihan, Réjane; Omilli, Francis; Deschamps, Stéphane; Pellerin, Isabelle

    2008-01-01

    Pre-B cell leukaemia transcription factors (PBXs) were originally identified as Hox cofactors, acting within transcriptional regulation complexes to regulate genetic programs during development. Increasing amount of evidence revealed that PBX function is not restricted to a partnership with Hox or homeodomain proteins. Indeed, PBXs are expressed throughout murine embryonic development and are involved in several developmental pathways including Hox-independent mechanisms. This review summarizes what is known about PBX partnerships and proposes to position PBXs as central developmental factors whose role consists of integrating transduction signals, in order to regulate gene expression programs during development.

  9. Differential utilization of the same reading frame in a Xenopus homeobox gene encodes two related proteins sharing the same DNA-binding specificity.

    PubMed Central

    Cho, K W; Goetz, J; Wright, C V; Fritz, A; Hardwicke, J; De Robertis, E M

    1988-01-01

    Xenopus XlHbox 1 produces two transcripts during early development. One encodes a long open reading frame (ORF) and the other a short ORF sharing the same homeodomain, but differing by an 82 amino acid domain at the amino terminus. The long protein amino terminus is conserved with many other homeodomain proteins, and its absence from the short protein could have functional consequences. Some viral genes also utilize a single ORF to encode transcription factors of antagonistic functions. The overall organization of the homologous genes in frog and man is similar, supporting the notion that both transcripts are of functional significance. Studies on XlHbox 1 function show that the region common to the long and short proteins has a sequence-specific DNA-binding activity, and that microinjection of specific antibodies into embryos results in the loss of structures derived from cells normally expressing XlHbox 1. Images PMID:2901347

  10. Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein.

    PubMed Central

    Ananthan, J; Baler, R; Morrissey, D; Zuo, J; Lan, Y; Weir, M; Voellmy, R

    1993-01-01

    Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins. Images PMID:8095092

  11. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  12. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  13. Protein

    USDA-ARS?s Scientific Manuscript database

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  14. Functional Specificity of a Hox Protein Mediated by the Recognition of Minor Groove Structure

    SciTech Connect

    Joshi,R.; Passner, J.; Rohs, R.; Jain, R.; Sosinksy, A.; Crickmore, M.; Jacob, V.; Aggarwal, A.; Honig, B.; et. al

    2007-01-01

    The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.

  15. Evolutionary and molecular analysis of Dof transcription factors identified a conserved motif for intercellular protein trafficking.

    PubMed

    Chen, Huan; Ahmad, Munawar; Rim, Yeonggil; Lucas, William J; Kim, Jae-Yean

    2013-06-01

    · Cell-to-cell trafficking of transcription factors (TFs) has been shown to play an important role in the regulation of plant developmental events, but the evolutionary relationship between cell-autonomous and noncell-autonomous (NCA) TFs remains elusive. · AtDof4.1, named INTERCELLULAR TRAFFICKING DOF 1 (ITD1), was chosen as a representative NCA member to explore this evolutionary relationship. Using domain structure-function analyses and swapping studies, we examined the cell-to-cell trafficking of plant-specific Dof TF family members across Arabidopsis and other species. · We identified a conserved intercellular trafficking motif (ITM) that is necessary and sufficient for selective cell-to-cell trafficking and can impart gain-of-function cell-to-cell movement capacity to an otherwise cell-autonomous TF. The functionality of related motifs from Dof members across the plant kingdom extended, surprisingly, to a unicellular alga that lacked plasmodesmata. By contrast, the algal homeodomain related to the NCA KNOX homeodomain was either inefficient or unable to impart such cell-to-cell movement function. · The Dof ITM appears to predate the evolution of selective plasmodesmal trafficking in the plant kingdom, which may well have acted as a molecular template for the evolution of Dof proteins as NCA TFs. However, the ability to efficiently traffic for KNOX homeodomain (HD) proteins may have been acquired during the evolution of early nonvascular plants.

  16. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  17. Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity.

    PubMed

    Hymus, Graham J; Cai, Suqin; Kohl, Elizabeth A; Holtan, Hans E; Marion, Colleen M; Tiwari, Shiv; Maszle, Don R; Lundgren, Marjorie R; Hong, Melissa C; Channa, Namitha; Loida, Paul; Thompson, Rebecca; Taylor, J Philip; Rice, Elena; Repetti, Peter P; Ratcliffe, Oliver J; Reuber, T Lynne; Creelman, Robert A

    2013-11-01

    Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.

  18. The mating type-specific homeodomain genes SXI1 alpha and SXI2a coordinately control uniparental mitochondrial inheritance in Cryptococcus neoformans.

    PubMed

    Yan, Zhun; Hull, Christina M; Sun, Sheng; Heitman, Joseph; Xu, Jianping

    2007-03-01

    In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type alpha (MATalpha) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type alpha (MATalpha)-specific gene SXI1a, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATalpha parent, and both the MATa and the MATalpha parents. In addition, progeny from same-sex mating between MATalpha strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1alpha and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.

  19. Strong expression of paired-like homeodomain transcription factor 1 (PITX1) is associated with a favorable outcome in human osteosarcoma.

    PubMed

    Kong, Gengbin; Liu, Zhaoyong; Wu, Kezhou; Zhang, Ying; Deng, Zhihua; Feng, Weili; Chen, Shubiao; Wang, Hu

    2015-09-01

    Paired-like homeodomain transcription factor 1 (PITX1) has been implicated as a tumor suppressor in various cancers. However, the biological and clinical significance of PITX1 in osteosarcoma has not been fully elucidated. Here, we studied the expression and clinical significance of PITX1 in 6 normal lower limb bone tissue specimens and 35 osteosarcoma tissue samples by immunohistochemistry. PITX1 was expressed in all normal tissues (6/6, 100 %) and in 85.7 % (30/35) of tumor tissues (P > 0.05). In addition, all normal tissue specimens showed high PITX1 expression (6/6, 100 %) while only 23.3 % (7/30) osteosarcoma tissue specimens had high PITX1 expression (P < 0.05). Patients with median overall survival (OS) >12 months had significantly higher PITX1 levels compared with those whose median OS was less than or equal to 12 months (P < 0.05 or 0.001). Furthermore, patients with lung metastasis had significantly lower PITX1 levels than patients without lung metastasis. In conclusion, PITX1 expression is downregulated in osteosarcoma and correlates with patient survival and lung metastasis.

  20. Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain

    PubMed Central

    Shimada, Naoto; Inami, Show; Sato, Shoma; Kitamoto, Toshihiro; Sakai, Takaomi

    2016-01-01

    Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. Here, we report that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. We identified that Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies. PMID:27853240

  1. Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain.

    PubMed

    Shimada, Naoto; Inami, Show; Sato, Shoma; Kitamoto, Toshihiro; Sakai, Takaomi

    2016-11-17

    Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. Here, we report that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. We identified that Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies.

  2. The Hairless Stem Phenotype of Cotton (Gossypium barbadense) Is Linked to a Copia-Like Retrotransposon Insertion in a Homeodomain-Leucine Zipper Gene (HD1)

    PubMed Central

    Ding, Mingquan; Ye, Wuwei; Lin, Lifeng; He, Shae; Du, Xiongming; Chen, Aiqun; Cao, Yuefen; Qin, Yuan; Yang, Fen; Jiang, Yurong; Zhang, Hua; Wang, Xiyin; Paterson, Andrew H.; Rong, Junkang

    2015-01-01

    Cotton (Gossypium) stem trichomes are mostly single cells that arise from stem epidermal cells. In this study, a homeodomain-leucine zipper gene (HD1) was found to cosegregate with the dominant trichome locus previously designated as T1 and mapped to chromosome 6. Characterization of HD1 orthologs revealed that the absence of stem trichomes in modern Gossypium barbadense varieties is linked to a large retrotransposon insertion in the ninth exon, 2565 bp downstream from the initial codon in the At subgenome HD1 gene (At-GbHD1). In both the At and Dt subgenomes, reduced transcription of GbHD1 genes is caused by this insertion. The disruption of At-HD1 further affects the expression of downstream GbMYB25 and GbHOX3 genes. Analyses of primitive cultivated accessions identified another retrotransposon insertion event in the sixth exon of At-GbHD1 that might predate the previously identified retrotransposon in modern varieties. Although both retrotransposon insertions results in similar phenotypic changes, the timing of these two retrotransposon insertion events fits well with our current understanding of the history of cotton speciation and dispersal. Taken together, the results of genetics mapping, gene expression and association analyses suggest that GbHD1 is an important component that controls stem trichome development and is a promising candidate gene for the T1 locus. The interspecific phenotypic difference in stem trichome traits also may be attributable to HD1 inactivation associated with retrotransposon insertion. PMID:26133897

  3. The Hairless Stem Phenotype of Cotton (Gossypium barbadense) Is Linked to a Copia-Like Retrotransposon Insertion in a Homeodomain-Leucine Zipper Gene (HD1).

    PubMed

    Ding, Mingquan; Ye, Wuwei; Lin, Lifeng; He, Shae; Du, Xiongming; Chen, Aiqun; Cao, Yuefen; Qin, Yuan; Yang, Fen; Jiang, Yurong; Zhang, Hua; Wang, Xiyin; Paterson, Andrew H; Rong, Junkang

    2015-09-01

    Cotton (Gossypium) stem trichomes are mostly single cells that arise from stem epidermal cells. In this study, a homeodomain-leucine zipper gene (HD1) was found to cosegregate with the dominant trichome locus previously designated as T1 and mapped to chromosome 6. Characterization of HD1 orthologs revealed that the absence of stem trichomes in modern Gossypium barbadense varieties is linked to a large retrotransposon insertion in the ninth exon, 2565 bp downstream from the initial codon in the At subgenome HD1 gene (At-GbHD1). In both the At and Dt subgenomes, reduced transcription of GbHD1 genes is caused by this insertion. The disruption of At-HD1 further affects the expression of downstream GbMYB25 and GbHOX3 genes. Analyses of primitive cultivated accessions identified another retrotransposon insertion event in the sixth exon of At-GbHD1 that might predate the previously identified retrotransposon in modern varieties. Although both retrotransposon insertions results in similar phenotypic changes, the timing of these two retrotransposon insertion events fits well with our current understanding of the history of cotton speciation and dispersal. Taken together, the results of genetics mapping, gene expression and association analyses suggest that GbHD1 is an important component that controls stem trichome development and is a promising candidate gene for the T1 locus. The interspecific phenotypic difference in stem trichome traits also may be attributable to HD1 inactivation associated with retrotransposon insertion.

  4. The Bicoid Class Homeodomain Factors ceh-36/OTX and unc-30/PITX Cooperate in C. elegans Embryonic Progenitor Cells to Regulate Robust Development

    PubMed Central

    Walton, Travis; Preston, Elicia; Nair, Gautham; Zacharias, Amanda L.; Raj, Arjun; Murray, John Isaac

    2015-01-01

    While many transcriptional regulators of pluripotent and terminally differentiated states have been identified, regulation of intermediate progenitor states is less well understood. Previous high throughput cellular resolution expression studies identified dozens of transcription factors with lineage-specific expression patterns in C. elegans embryos that could regulate progenitor identity. In this study we identified a broad embryonic role for the C. elegans OTX transcription factor ceh-36, which was previously shown to be required for the terminal specification of four neurons. ceh-36 is expressed in progenitors of over 30% of embryonic cells, yet is not required for embryonic viability. Quantitative phenotyping by computational analysis of time-lapse movies of ceh-36 mutant embryos identified cell cycle or cell migration defects in over 100 of these cells, but most defects were low-penetrance, suggesting redundancy. Expression of ceh-36 partially overlaps with that of the PITX transcription factor unc-30. unc-30 single mutants are viable but loss of both ceh-36 and unc-30 causes 100% lethality, and double mutants have significantly higher frequencies of cellular developmental defects in the cells where their expression normally overlaps. These factors are also required for robust expression of the downstream developmental regulator mls-2/HMX. This work provides the first example of genetic redundancy between the related yet evolutionarily distant OTX and PITX families of bicoid class homeodomain factors and demonstrates the power of quantitative developmental phenotyping in C. elegans to identify developmental regulators acting in progenitor cells. PMID:25738873

  5. The NK-2 class homeodomain factor CEH-51 and the T-box factor TBX-35 have overlapping function in C. elegans mesoderm development.

    PubMed

    Broitman-Maduro, Gina; Owraghi, Melissa; Hung, Wendy W K; Kuntz, Steven; Sternberg, Paul W; Maduro, Morris F

    2009-08-01

    The C. elegans MS blastomere, born at the 7-cell stage of embryogenesis, generates primarily mesodermal cell types, including pharynx cells, body muscles and coelomocytes. A presumptive null mutation in the T-box factor gene tbx-35, a target of the MED-1 and MED-2 divergent GATA factors, was previously found to result in a profound decrease in the production of MS-derived tissues, although the tbx-35(-) embryonic arrest phenotype was variable. We report here that the NK-2 class homeobox gene ceh-51 is a direct target of TBX-35 and at least one other factor, and that CEH-51 and TBX-35 share functions. Embryos homozygous for a ceh-51 null mutation arrest as larvae with pharynx and muscle defects, although these tissues appear to be specified correctly. Loss of tbx-35 and ceh-51 together results in a synergistic phenotype resembling loss of med-1 and med-2. Overexpression of ceh-51 causes embryonic arrest and generation of ectopic body muscle and coelomocytes. Our data show that TBX-35 and CEH-51 have overlapping function in MS lineage development. As T-box regulators and NK-2 homeodomain factors are both important for heart development in Drosophila and vertebrates, our results suggest that these regulators function in a similar manner in C. elegans to specify a major precursor of mesoderm.

  6. The Bicoid class homeodomain factors ceh-36/OTX and unc-30/PITX cooperate in C. elegans embryonic progenitor cells to regulate robust development.

    PubMed

    Walton, Travis; Preston, Elicia; Nair, Gautham; Zacharias, Amanda L; Raj, Arjun; Murray, John Isaac

    2015-03-01

    While many transcriptional regulators of pluripotent and terminally differentiated states have been identified, regulation of intermediate progenitor states is less well understood. Previous high throughput cellular resolution expression studies identified dozens of transcription factors with lineage-specific expression patterns in C. elegans embryos that could regulate progenitor identity. In this study we identified a broad embryonic role for the C. elegans OTX transcription factor ceh-36, which was previously shown to be required for the terminal specification of four neurons. ceh-36 is expressed in progenitors of over 30% of embryonic cells, yet is not required for embryonic viability. Quantitative phenotyping by computational analysis of time-lapse movies of ceh-36 mutant embryos identified cell cycle or cell migration defects in over 100 of these cells, but most defects were low-penetrance, suggesting redundancy. Expression of ceh-36 partially overlaps with that of the PITX transcription factor unc-30. unc-30 single mutants are viable but loss of both ceh-36 and unc-30 causes 100% lethality, and double mutants have significantly higher frequencies of cellular developmental defects in the cells where their expression normally overlaps. These factors are also required for robust expression of the downstream developmental regulator mls-2/HMX. This work provides the first example of genetic redundancy between the related yet evolutionarily distant OTX and PITX families of bicoid class homeodomain factors and demonstrates the power of quantitative developmental phenotyping in C. elegans to identify developmental regulators acting in progenitor cells.

  7. Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity

    PubMed Central

    Kohl, Elizabeth A.; Tiwari, Shiv; Lundgren, Marjorie R.; Channa, Namitha; Creelman, Robert A.

    2013-01-01

    Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines. PMID:24006420

  8. Ex vivo expansion of normal and chronic myeloid leukemic stem cells without functional alteration using a NUP98HOXA10homeodomain fusion gene

    PubMed Central

    Sloma, I; Imren, S; Beer, P A; Zhao, Y; Lecault, V; Leung, D; Raghuram, K; Brimacombe, C; Lambie, K; Piret, J; Hansen, C; Humphries, R K; Eaves, C J

    2013-01-01

    HOX genes have been implicated as regulators of normal and leukemic stem cell functionality, but the extent to which these activities are linked is poorly understood. Previous studies revealed that transduction of primitive mouse hematopoietic cells with a NUP98HOXA10homeodomain (NA10HD) fusion gene enables a subsequent rapid and marked expansion in vitro of hematopoietic stem cell numbers without causing their transformation or deregulated expansion in vivo. To determine whether forced expression of NA10HD in primitive human cells would have a similar effect, we compared the number of long-term culture-initiating cells (LTC-ICs) present in cultures of lenti-NA10HD versus control virus-transduced CD34+ cells originally isolated from human cord blood and chronic phase (CP) chronic myeloid leukemia (CML) patients. We found that NA10HD greatly increases outputs of both normal and Ph+/BCR-ABL+ LTC-ICs, and this effect is particularly pronounced in cultures containing growth factor-producing feeders. Interestingly, NA10HD did not affect the initial cell cycle kinetics of the transduced cells nor their subsequent differentiation. Moreover, immunodeficient mice repopulated with NA10HD-transduced CP-CML cells for more than 8 months showed no evidence of altered behavior. Thus, NA10HD provides a novel tool to enhance both normal and CP-CML stem cell expansion in vitro, without apparently altering other properties. PMID:22868969

  9. Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning.

    PubMed

    Waskiewicz, A J; Rikhof, H A; Hernandez, R E; Moens, C B

    2001-11-01

    Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr(-) phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

  10. The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity.

    PubMed

    Berthelsen, J; Zappavigna, V; Ferretti, E; Mavilio, F; Blasi, F

    1998-03-02

    The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

  11. A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo.

    PubMed Central

    Simeone, A; Acampora, D; Mallamaci, A; Stornaiuolo, A; D'Apice, M R; Nigro, V; Boncinelli, E

    1993-01-01

    We studied the expression of two vertebrate homeobox genes, Otx1 and Otx2, related to orthodenticle, a gene expressed in the developing head of Drosophila. Both genes are expressed in restricted regions of the developing rostral brain including the presumptive cerebral cortex and olfactory bulbs. The expression patterns of the two genes in diencephalon suggest that they both have a role in establishing the boundary between presumptive dorsal and ventral thalamus. They are also expressed in regions of the developing olfactory, auricolar and ocular system, including the covering of the optic nerve. Otx1 expression is detectable from day 8 of gestation in telencephalic, diencephalic and mesencephalic regions. From day 10.5 of gestation its expression extends to some metencephalic areas. Otx2 appears to be already expressed in the epiblast of prestreak embryos. It persists in the entire embryonic ectoderm for some time after the onset of gastrulation. In midstreak embryos its expression appears progressively restricted to the anterior embryonic ectoderm corresponding to presumptive fore- and mid-brain. In early midgestation embryos it is expressed in telencephalic, diencephalic and mesencephalic regions but from day 11.75 of gestation its expression disappears from dorsal telencephalon and is confined to diencephalic and mesencephalic regions. Otx2 is one of the earliest genes expressed in the epiblast and immediately afterwards is expressed in anterior neuroectoderm, demarcating rostral brain regions even before headfold formation. Its gene product contains a homeodomain of the bicoid class and is able to recognize and transactivate a bicoid target sequence. Images PMID:8101484

  12. The LIM homeodomain transcription factor LHX6: a transcriptional repressor that interacts with pituitary homeobox 2 (PITX2) to regulate odontogenesis.

    PubMed

    Zhang, Zichao; Gutierrez, Diana; Li, Xiao; Bidlack, Felicitas; Cao, Huojun; Wang, Jianbo; Andrade, Kelsey; Margolis, Henry C; Amendt, Brad A

    2013-01-25

    LHX6 is a LIM-homeobox transcription factor expressed during embryogenesis; however, the molecular mechanisms regulating LHX6 transcriptional activities are unknown. LHX6 and the PITX2 homeodomain transcription factor have overlapping expression patterns during tooth and craniofacial development, and in this report, we demonstrate new transcriptional mechanisms for these factors. PITX2 and LHX6 are co-expressed in the oral and dental epithelium and epithelial cell lines. Lhx6 expression is increased in Pitx2c transgenic mice and decreased in Pitx2 null mice. PITX2 activates endogenous Lhx6 expression and the Lhx6 promoter, whereas LHX6 represses its promoter activity. Chromatin immunoprecipitation experiments reveal endogenous PITX2 binding to the Lhx6 promoter. LHX6 directly interacts with PITX2 to inhibit PITX2 transcriptional activities and activation of multiple promoters. Bimolecular fluorescence complementation assays reveal an LHX6·PITX2 nuclear interaction in living cells. LHX6 has a dominant repressive effect on the PITX2 synergistic activation with LEF-1 and β-catenin co-factors. Thus, LHX6 acts as a transcriptional repressor and represses the expression of several genes involved in odontogenesis. We have identified specific defects in incisor, molar, mandible, bone, and root development and late stage enamel formation in Lhx6 null mice. Amelogenin and ameloblastin expression is reduced and/or delayed in the Lhx6 null mice, potentially resulting from defects in dentin deposition and ameloblast differentiation. Our results demonstrate that LHX6 regulates cell proliferation in the cervical loop and promotes cell differentiation in the anterior region of the incisor. We demonstrate new molecular mechanisms for LHX6 and an interaction with PITX2 for normal craniofacial and tooth development.

  13. TTF1, a homeodomain containing transcription factor, contributes to regulating periodic oscillations in GnRH gene expression

    PubMed Central

    Matagne, Valerie; Kim, Jae Geun; Ryu, Byung Jun; Hur, Min Kyu; Kim, Min Sung; Kim, Kyungjin; Park, Byong Seo; Damante, Giuseppe; Smiley, Gregory; Lee, Byung Ju; Ojeda, Sergio R.

    2012-01-01

    Thyroid transcription factor 1 (TTF1), a member of the NK family of transcription factors required for basal forebrain morphogenesis, functions in the postnatal hypothalamus as a transcriptional regulator of genes encoding neuromodulators and hypophysiotrophic peptides. One of these peptides is gonadotropin-releasing hormone (GnRH). Here we show that Ttf1 mRNA abundance vary in a diurnal and melatonin-dependent fashion in the preoptic area (POA) of the rat, with maximal Ttf1 expression attained during the dark phase of the light/dark cycle, preceding the nocturnal peak in GnRH mRNA content. GnRH promoter activity oscillates in a circadian manner in GT1-7 cells, and this pattern is enhanced by TTF1 and blunted by siRNA-mediated Ttf1 gene silencing. TTF1 trans-activates GnRH transcription by binding to two sites in the GnRH promoter. Rat GnRH neurons in situ contain key proteins components of the positive (BMAL1, CLOCK) and negative (PER1) limbs of the circadian oscillator, and these proteins repress Ttf1 promoter activity in vitro. In contrast, Ttf1 transcription is activated by CRY1, a clock component required for circadian rhythmicity. In turn, TTF1 represses transcription of Rev-erbα, a heme receptor that controls circadian transcription within the positive limb of the circadian oscillator. These findings suggest that TTF1 is a component of the molecular machinery controlling circadian oscillations in GnRH gene transcription. PMID:22356123

  14. Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.

    PubMed Central

    Gregory, S L; Kortschak, R D; Kalionis, B; Saint, R

    1996-01-01

    We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others. PMID:8622680

  15. Genome-wide identification and expression profile of homeodomain-leucine zipper Class I gene family in Cucumis sativus.

    PubMed

    Liu, Wei; Fu, Rao; Li, Qiang; Li, Jing; Wang, Lina; Ren, Zhonghai

    2013-12-01

    The HD-Zip proteins comprise one of the largest families of transcription factors in plants. HD-Zip genes have been grouped into four different classes: HD-Zip I to IV. In this study, we described the identification and structural characterization of Class I HD-Zip genes in cucumber. A complete set of 13 HD-Zip I genes were identified in the cucumber genome using Blast search tools and phylogeny. The cucumber HD-Zip I family contained a smaller number of identified genes compared to other higher plants such as Arabidopsis and maize due to the absence of recent gene duplication events. Chromosomal location of these genes revealed that they are distributed unevenly across 5 of 7 chromosomes. Tissue-specific expression profiles showed that 13 cucumber HD-Zip I genes were expressed in at least one of the tissues, which suggested that cucumber HD-Zip I genes took part in many cellular processes. The transcript abundance level analysis during abiotic stress conditions (NaCl, ABA and low temperature treatments) identified a group of HD-Zip I genes that responded to one or more treatments.

  16. The Homeodomain Transcription Factor Cdx1 Does Not Behave as an Oncogene in Normal Mouse Intestine1

    PubMed Central

    Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

    2008-01-01

    The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium. PMID:18231635

  17. Affinity regression predicts the recognition code of nucleic acid binding proteins

    PubMed Central

    Pelossof, Raphael; Singh, Irtisha; Yang, Julie L.; Weirauch, Matthew T.; Hughes, Timothy R.; Leslie, Christina S.

    2016-01-01

    Predicting the affinity profiles of nucleic acid-binding proteins directly from the protein sequence is a major unsolved problem. We present a statistical approach for learning the recognition code of a family of transcription factors (TFs) or RNA-binding proteins (RBPs) from high-throughput binding assays. Our method, called affinity regression, trains on protein binding microarray (PBM) or RNA compete experiments to learn an interaction model between proteins and nucleic acids, using only protein domain and probe sequences as inputs. By training on mouse homeodomain PBM profiles, our model correctly identifies residues that confer DNA-binding specificity and accurately predicts binding motifs for an independent set of divergent homeodomains. Similarly, learning from RNA compete profiles for diverse RBPs, our model can predict the binding affinities of held-out proteins and identify key RNA-binding residues. More broadly, we envision applying our method to model and predict biological interactions in any setting where there is a high-throughput ‘affinity’ readout. PMID:26571099

  18. A novel Glycine soja homeodomain-leucine zipper (HD-Zip) I gene, Gshdz4, positively regulates bicarbonate tolerance and responds to osmotic stress in Arabidopsis.

    PubMed

    Cao, Lei; Yu, Yang; DuanMu, Huizi; Chen, Chao; Duan, Xiangbo; Zhu, Pinghui; Chen, Ranran; Li, Qiang; Zhu, Yanming; Ding, Xiaodong

    2016-08-24

    Wild soybean (Glycine soja) is a highly adaptive plant species which can grow well in saline-alkaline soils. In soybean genome, there exist about 140 HD-Zip (Homeodomain-leucine Zipper) genes. HD-Zip transcription factor family is one of the largest plant specific superfamilies and plays important roles in response to abiotic stresses. Although HD-Zip transcription factors have been broadly reported to be involved in plant resistance to abiotic stresses like salt and drought, their roles in response to bicarbonate stress is largely unknown. From our previous transcriptome profile analysis of wild soybean treated by 50 mM NaHCO3, we identified an HD-Zip gene (Gshdz4) which showed high response to the alkaline stress. Our result of qRT-PCR showed that the expression of Gshdz4 was induced by alkaline stress (NaHCO3) in both leaves and roots of wild soybean. Overexpression of Gshdz4 in Arabidopsis resulted in enhanced tolerance to NaHCO3 and KHCO3 during the process of plant growth and development. However, the growths of transgenic and WT plants were not significantly different on the medium with high pH adjusted by KOH, implicating Gshdz4 is only responsible for resisting HCO3 (-) but not high pH. The transgenic plants had less MDA contents but higher POD activities and chlorophyll contents than the WT plants. Moreover, the transcript levels of stress-related genes, such as NADP-ME, H (+) -Ppase, RD29B and KIN1 were increased with greater extent in the transgenic plants than the wild plants. On the contrary, Gshdz4 overexpression lines were much sensitive to osmotic stress at seed germination and stocking stages compared to the wild plants. We revealed that the important and special roles of Gshdz4 in enhancing bicarbonate tolerance and responding to osmotic stress. It is the first time to elucidate these novel functions of HD-ZIP transcription factors. All the evidences broaden our understanding of functions of HD-Zip family and provide clues for uncovering the

  19. A survey of conservation of sea spider and Drosophila Hox protein activities.

    PubMed

    Saadaoui, Mehdi; Litim-Mecheri, Isma; Macchi, Meiggie; Graba, Yacine; Maurel-Zaffran, Corinne

    2015-11-01

    Hox proteins have well-established functions in development and evolution, controlling the final morphology of bilaterian animals. The common phylogenetic origin of Hox proteins and the associated evolutionary diversification of protein sequences provide a unique framework to explore the relationship between changes in protein sequence and function. In this study, we aimed at questioning how sequence variation within arthropod Hox proteins influences function. This was achieved by exploring the functional impact of sequence conservation/divergence of the Hox genes, labial, Sex comb reduced, Deformed, Ultrabithorax and abdominalA from two distant arthropods, the sea spider and the well-studied Drosophila. Results highlight a correlation between sequence conservation within the homeodomain and the degree of functional conservation, and identify a novel functional domain in the Labial protein.

  20. ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation.

    PubMed

    Karagianni, Panagiota; Amazit, Larbi; Qin, Jun; Wong, Jiemin

    2008-01-01

    Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

  1. Expression of Meis and Pbx genes and their protein products in the developing telencephalon: implications for regional differentiation.

    PubMed

    Toresson, H; Parmar, M; Campbell, K

    2000-06-01

    The Meis and Pbx genes encode for homeodomain proteins of the TALE class and have been shown to act as co-factors for other homeodomain transcription factors (Mann and Affolter, 1998. Curr. Opin. Genet. Dev. 8, 423-429). We have studied the expression of these genes in the mouse telencephalon and found that Meis1 and Meis2 display region-specific patterns of expression from embryonic day (E)10.5 until birth, defining distinct subterritories in the developing telencephalon. The expression of the Meis genes and their proteins is highest in the subventricular zone (SVZ) and mantle regions of the ventral telencephalon. Compared to the Meis genes, Pbx genes show a broader expression within the telencephalon. However, as is the case in Drosophila (Rieckhof et al., 1997. Cell 91, 171-183; Kurrant et al., 1998. Development 125, 1037-1048; Pai et al., 1998. Genes Dev. 12, 435-446), nuclear localized PBX proteins were found to correlate highly with Meis expression. In addition, DLX proteins co-localize with nuclear PBX in distinct regions of the ventral telencephalon.

  2. The Arabidopsis thaliana vernalization response requires a polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3.

    PubMed

    Wood, Craig C; Robertson, Masumi; Tanner, Greg; Peacock, W James; Dennis, Elizabeth S; Helliwell, Chris A

    2006-09-26

    In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION INSENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression.

  3. Computational design of co-assembling protein-DNA nanowires.

    PubMed

    Mou, Yun; Yu, Jiun-Yann; Wannier, Timothy M; Guo, Chin-Lin; Mayo, Stephen L

    2015-09-10

    Biomolecular self-assemblies are of great interest to nanotechnologists because of their functional versatility and their biocompatibility. Over the past decade, sophisticated single-component nanostructures composed exclusively of nucleic acids, peptides and proteins have been reported, and these nanostructures have been used in a wide range of applications, from drug delivery to molecular computing. Despite these successes, the development of hybrid co-assemblies of nucleic acids and proteins has remained elusive. Here we use computational protein design to create a protein-DNA co-assembling nanomaterial whose assembly is driven via non-covalent interactions. To achieve this, a homodimerization interface is engineered onto the Drosophila Engrailed homeodomain (ENH), allowing the dimerized protein complex to bind to two double-stranded DNA (dsDNA) molecules. By varying the arrangement of protein-binding sites on the dsDNA, an irregular bulk nanoparticle or a nanowire with single-molecule width can be spontaneously formed by mixing the protein and dsDNA building blocks. We characterize the protein-DNA nanowire using fluorescence microscopy, atomic force microscopy and X-ray crystallography, confirming that the nanowire is formed via the proposed mechanism. This work lays the foundation for the development of new classes of protein-DNA hybrid materials. Further applications can be explored by incorporating DNA origami, DNA aptamers and/or peptide epitopes into the protein-DNA framework presented here.

  4. Computational design of co-assembling protein-DNA nanowires

    NASA Astrophysics Data System (ADS)

    Mou, Yun; Yu, Jiun-Yann; Wannier, Timothy M.; Guo, Chin-Lin; Mayo, Stephen L.

    2015-09-01

    Biomolecular self-assemblies are of great interest to nanotechnologists because of their functional versatility and their biocompatibility. Over the past decade, sophisticated single-component nanostructures composed exclusively of nucleic acids, peptides and proteins have been reported, and these nanostructures have been used in a wide range of applications, from drug delivery to molecular computing. Despite these successes, the development of hybrid co-assemblies of nucleic acids and proteins has remained elusive. Here we use computational protein design to create a protein-DNA co-assembling nanomaterial whose assembly is driven via non-covalent interactions. To achieve this, a homodimerization interface is engineered onto the Drosophila Engrailed homeodomain (ENH), allowing the dimerized protein complex to bind to two double-stranded DNA (dsDNA) molecules. By varying the arrangement of protein-binding sites on the dsDNA, an irregular bulk nanoparticle or a nanowire with single-molecule width can be spontaneously formed by mixing the protein and dsDNA building blocks. We characterize the protein-DNA nanowire using fluorescence microscopy, atomic force microscopy and X-ray crystallography, confirming that the nanowire is formed via the proposed mechanism. This work lays the foundation for the development of new classes of protein-DNA hybrid materials. Further applications can be explored by incorporating DNA origami, DNA aptamers and/or peptide epitopes into the protein-DNA framework presented here.

  5. Imaging the Localized Protein Interactions Between Pit-1 and the CCAAT/Enhancer Binding Protein α in the Living Pituitary Cell Nucleus

    PubMed Central

    Day, Richard N.; Voss, Ty C.; Enwright, John F.; Booker, Cynthia F.; Periasamy, Ammasi; Schaufele, Fred

    2010-01-01

    The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein α (C/EBPα) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPα was concentrated in regions of centromeric heterochromatin in pituitary GHFT1–5 cells and that coexpressed Pit-1 redistributed C/EBPα to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPα was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBPα transactivation domain disrupted the redistribution of C/EBPα by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPα, and the truncated C/EBPα still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPα that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPα to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBPα are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBPα at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression. PMID:12554785

  6. The Paired-box Homeodomain Transcription Factor Pax6 Binds to the Upstream Region of the TRAP Gene Promoter and Suppresses Receptor Activator of NF-κB Ligand (RANKL)-induced Osteoclast Differentiation*

    PubMed Central

    Kogawa, Masakazu; Hisatake, Koji; Atkins, Gerald J.; Findlay, David M.; Enoki, Yuichiro; Sato, Tsuyoshi; Gray, Peter C.; Kanesaki-Yatsuka, Yukiko; Anderson, Paul H.; Wada, Seiki; Kato, Naoki; Fukuda, Aya; Katayama, Shigehiro; Tsujimoto, Masafumi; Yoda, Tetsuya; Suda, Tatsuo; Okazaki, Yasushi; Matsumoto, Masahito

    2013-01-01

    Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption. PMID:23990468

  7. The zinc finger proteins Pannier and GATA4 function as cardiogenic factors in Drosophila.

    PubMed

    Gajewski, K; Fossett, N; Molkentin, J D; Schulz, R A

    1999-12-01

    The regulation of cardiac gene expression by GATA zinc finger transcription factors is well documented in vertebrates. However, genetic studies in mice have failed to demonstrate a function for these proteins in cardiomyocyte specification. In Drosophila, the existence of a cardiogenic GATA factor has been implicated through the analysis of a cardial cell enhancer of the muscle differentiation gene D-mef2. We show that the GATA gene pannier is expressed in the dorsal mesoderm and required for cardial cell formation while repressing a pericardial cell fate. Ectopic expression of Pannier results in cardial cell overproduction, while co-expression of Pannier and the homeodomain protein Tinman synergistically activate cardiac gene expression and induce cardial cells. The related GATA4 protein of mice likewise functions as a cardiogenic factor in Drosophila, demonstrating an evolutionarily conserved function between Pannier and GATA4 in heart development.

  8. An integrated genomic analysis of Tudor domain-containing proteins identifies PHD finger protein 20-like 1 (PHF20L1) as a candidate oncogene in breast cancer.

    PubMed

    Jiang, Yuanyuan; Liu, Lanxin; Shan, Wenqi; Yang, Zeng-Quan

    2016-02-01

    Tudor domain-containing proteins (TDRDs), which recognize and bind to methyl-lysine/arginine residues on histones and non-histone proteins, play critical roles in regulating chromatin architecture, transcription, genomic stability, and RNA metabolism. Dysregulation of several TDRDs have been observed in various types of cancer. However, neither the genomic landscape nor clinical significance of TDRDs in breast cancer has been explored comprehensively. Here, we performed an integrated genomic and transcriptomic analysis of 41 TDRD genes in breast cancer (TCGA and METABRIC datasets) and identified associations among recurrent copy number alterations, gene expressions, clinicopathological features, and survival of patients. Among seven TDRDs that had the highest frequency (>10%) of gene amplification, the plant homeodomain finger protein 20-like 1 (PHF20L1) was the most commonly amplified (17.62%) TDRD gene in TCGA breast cancers. Different subtypes of breast cancer had different patterns of copy number and expression for each TDRD. Notably, amplification and overexpression of PHF20L1 were more prevalent in aggressive basal-like and Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. Furthermore, knockdown of PHF20L1 inhibited cell proliferation in PHF20L1-amplified breast cancer cell lines. PHF20L1 protein contains N-terminal Tudor and C-terminal plant homeodomain domains. Detailed characterization of PHF20L1 in breast cancer revealed that the Tudor domain likely plays a critical role in promoting cancer. Mechanistically, PHF20L1 might participate in regulating DNA methylation by stabilizing DNA methyltransferase 1 (DNMT1) protein in breast cancer. Thus, our results demonstrated the oncogenic potential of PHF20L1 and its association with poor prognostic parameters in breast cancer. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. The PBX-regulating protein PREP1 is present in different PBX-complexed forms in mouse.

    PubMed

    Ferretti, E; Schulz, H; Talarico, D; Blasi, F; Berthelsen, J

    1999-05-01

    Human PREP1, a novel homeodomain protein of the TALE super-family, forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters (Berthelsen, J., Zappavigna, V., Ferretti, E., Mavilio, F., Blasi, F. , 1998b. The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperatity. EMBO J. 17, 1434-1445; Berthelsen, J., Zappavigna, V., Mavilio, F., Blasi, F., 1998c. Prep1, a novel functional partner of Pbx proteins. EMBO J. 17, 1423-1433). Here we demonstrate the presence of DNA-binding PREP1-PBX complexes also in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. We report the cloning and expression domain of murine Prep1 gene. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. We further demonstrate the presence of murine Prep1 mRNA and protein, and of different DNA-binding PREP1-PBX complexes, in mouse embryos from at least 9.5 days p.c. Moreover, we show that PREP1 is present in all embryonic tissues from at least 7.5-17.5 days p.c with a predominantly nuclear staining. PREP1 is able to super-activate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and we show that all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role of PREP1 as a regulator of PBX and HOXB-1 proteins activity during development.

  10. Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

    PubMed

    Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

  11. Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

    PubMed Central

    Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

  12. Emx2 homeodomain transcription factor interacts with eukaryotic translation initiation factor 4E (eIF4E) in the axons of olfactory sensory neurons.

    PubMed

    Nédélec, Stéphane; Foucher, Isabelle; Brunet, Isabelle; Bouillot, Colette; Prochiantz, Alain; Trembleau, Alain

    2004-07-20

    We report that Emx2 homeogene is expressed at the mRNA and protein levels in the adult mouse olfactory neuroepithelium. As expected for a transcription factor, Emx2 is present in the nucleus of immature and mature olfactory sensory neurons. However, the protein is also detected in the axonal compartment of these neurons, both in the olfactory mucosa axon bundles and in axon terminals within the olfactory bulb. Emx2 axonal staining is heterogeneous, suggesting an association with particles. Subcellular fractionations of olfactory bulb synaptosomes, combined with chemical lesions of olfactory neurons, confirm the presence of Emx2 in axon terminals. Significant amounts of Emx2 protein cosediment with high density synaptosomal subfractions containing eukaryotic translation initiation factor 4E (eIF4E). Nonionic detergents and RNase treatments failed to detach eIF4E and Emx2 from these high-density fractions enriched in vesicles and granular structures. In addition, Emx2 and eIF4E can be coimmunoprecipitated from olfactory mucosa and bulb extracts and interact directly, as demonstrated in pull-down experiments. Emx2 axonal localization, association with high-density particles and interaction with eIF4E strongly suggest that this transcription factor has new nonnuclear functions most probably related to the local control of protein translation in the olfactory sensory neuron axons. Finally, we show that two other brain-expressed homeoproteins, Otx2 and Engrailed 2, also bind eIF4E, indicating that several homeoproteins may modulate eIF4E functions in the developing and adult nervous system.

  13. Coordinate regulation of gene expression in the C. elegans excretory cell by the POU domain protein CEH-6.

    PubMed

    Armstrong, Kristin R; Chamberlin, Helen M

    2010-01-01

    Excretory renal organs are critical in animals for osmoregulation and the elimination of waste. Renal organs across a range of species exhibit cellular and molecular similarities. For example, class III POU-homeodomain transcription factors are expressed in the renal organs of many invertebrates and vertebrates. However, the functional role for these factors is not well characterized. To better understand the role of class III POU-homeodomain proteins in animal excretory systems, we have characterized a set of genes expressed in the Caenorhabditis elegans excretory cell, and determined their regulation by the POU-III transcription factor CEH-6. Our molecular and biochemical studies show that CEH-6 regulates a subset of genes expressed in the excretory cell. Additionally, we find that the CEH-6-dependent genes share two molecular features: they contain at least one octamer regulatory element and they encode for transport and channel proteins. This work suggests that a role for POU-III factors in renal organs is to coordinate the expression of a set of functionally related genes.

  14. Hyperglycemia triggers HIPK2 protein degradation

    PubMed Central

    Granato, Marisa; Cuomo, Laura; Pistritto, Giuseppa; Cirone, Mara; D'Orazi, Gabriella

    2017-01-01

    Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies. PMID:27901482

  15. A Comprehensive Analysis of MicroProteins Reveals Their Potentially Widespread Mechanism of Transcriptional Regulation1[W

    PubMed Central

    Magnani, Enrico; de Klein, Niek; Nam, Hye-In; Kim, Jung-Gun; Pham, Kimberly; Fiume, Elisa; Mudgett, Mary Beth; Rhee, Seung Yon

    2014-01-01

    Truncated transcription factor-like proteins called microProteins (miPs) can modulate transcription factor activities, thereby increasing transcriptional regulatory complexity. To understand their prevalence, evolution, and function, we predicted over 400 genes that encode putative miPs from Arabidopsis (Arabidopsis thaliana) using a bioinformatics pipeline and validated two novel miPs involved in flowering time and response to abiotic and biotic stress. We provide an evolutionary perspective for a class of miPs targeting homeodomain transcription factors in plants and metazoans. We identify domain loss as one mechanism of miP evolution and suggest the possible roles of miPs on the evolution of their target transcription factors. Overall, we reveal a prominent layer of transcriptional regulation by miPs, show pervasiveness of such proteins both within and across genomes, and provide a framework for studying their function and evolution. PMID:24616380

  16. The TALE face of Hox proteins in animal evolution

    PubMed Central

    Merabet, Samir; Galliot, Brigitte

    2015-01-01

    Hox genes are major regulators of embryonic development. One of their most conserved functions is to coordinate the formation of specific body structures along the anterior-posterior (AP) axis in Bilateria. This architectural role was at the basis of several morphological innovations across bilaterian evolution. In this review, we traced the origin of the Hox patterning system by considering the partnership with PBC and Meis proteins. PBC and Meis belong to the TALE-class of homeodomain-containing transcription factors and act as generic cofactors of Hox proteins for AP axis patterning in Bilateria. Recent data indicate that Hox proteins acquired the ability to interact with their TALE partners in the last common ancestor of Bilateria and Cnidaria. These interactions relied initially on a short peptide motif called hexapeptide (HX), which is present in Hox and non-Hox protein families. Remarkably, Hox proteins can also recruit the TALE cofactors by using specific PBC Interaction Motifs (SPIMs). We describe how a functional Hox/TALE patterning system emerged in eumetazoans through the acquisition of SPIMs. We anticipate that interaction flexibility could be found in other patterning systems, being at the heart of the astonishing morphological diversity observed in the animal kingdom. PMID:26347770

  17. Protein phosphatase-1 modulates the function of Pax-6, a transcription factor controlling brain and eye development.

    PubMed

    Yan, Qin; Liu, Wen-Bin; Qin, Jichao; Liu, Jinping; Chen, He-Ge; Huang, Xiaoqin; Chen, Lili; Sun, Shuming; Deng, Mi; Gong, Lili; Li, Yong; Zhang, Lan; Liu, Yan; Feng, Hao; Xiao, Yamei; Liu, Yun; Li, David W-C

    2007-05-11

    Pax-6 is an evolutionarily conserved transcription factor and acts high up in the regulatory hierarchy controlling eye and brain development in humans, mice, zebrafish, and Drosophila. Previous studies have shown that Pax-6 is a phosphoprotein, and its phosphorylation by ERK, p38, and homeodomain-interacting protein kinase 2 greatly enhances its transactivation activity. However, the protein phosphatases responsible for the dephosphorylation of Pax-6 remain unknown. Here, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 is a major phosphatase that directly dephosphorylates Pax-6. First, purified protein phosphatase-1 directly dephosphorylates Pax-6 in vitro. Second, immunoprecipitation-linked Western blot revealed that both protein phosphatase-1alpha and protein phosphatase-1beta interact with Pax-6. Third, overexpression of protein phosphatase-1 in human lens epithelial cells leads to dephosphorylation of Pax-6. Finally, inhibition of protein phosphatase-1 activity by calyculin A or knockdown of protein phosphatase-1alpha and protein phosphatase-1beta by RNA interference leads to enhanced phosphorylation of Pax-6. Moreover, our results also demonstrate that dephosphorylation of Pax-6 by protein phosphatase-1 significantly modulates its function in regulating expression of both exogenous and endogenous genes. These results demonstrate that protein phosphatase 1 acts as a major phosphatase to dephosphorylate Pax-6 and modulate its function.

  18. Structural basis of plant homeodomain finger 6 (PHF6) recognition by the retinoblastoma binding protein 4 (RBBP4) component of the nucleosome remodeling and deacetylase (NuRD) complex.

    PubMed

    Liu, Zhonghua; Li, Fudong; Zhang, Beibei; Li, Sai; Wu, Jihui; Shi, Yunyu

    2015-03-06

    The NuRD complex is a conserved transcriptional coregulator that contains both chromatin-remodeling and histone deacetylase activities. Mutations of PHF6 are found in patients with Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, or acute myeloid leukemia. Recently, PHF6 was identified to interact with the NuRD complex, and this interaction is mediated by the RBBP4 component. However, little is known about the molecular basis for the interaction. Here, we present the crystal structure of the complex of the NuRD subunit RBBP4 bound to the PHF6 peptide (residues 162-170). The PHF6 peptide binds to the top surface of the RBBP4 β-propeller. A pair of positively charged residues of the PHF6 peptide insert into the negatively charged pocket of RBBP4, which is critical for the interaction between PHF6 and RBBP4. Corresponding PHF6 mutants impair this interaction in vitro and in vivo. Structural comparison shows that the PHF6-binding pocket overlaps with FOG1 and histone H3 on RBBP4/Nurf55, but it is distinct from the pocket recognizing histone H4, Su(z)12, and MTA1. We further show that the middle disordered region (residues 145-207, containing the RBBP4-binding motif) is sufficient for the transcriptional repression mediated by PHF6 on the GAL4 reporter, and knockdown of RBBP4 diminished the PHF6-mediated repression. Our RBBP4-PHF6 complex structure provides insights into the molecular basis of PHF6-NuRD complex interaction and implicates a role for PHF6 in chromatin structure modulation and gene regulation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Comparative analysis of ATRX, a chromatin remodeling protein.

    PubMed

    Park, Daniel J; Pask, Andrew J; Huynh, Kim; Renfree, Marilyn B; Harley, Vincent R; Graves, Jennifer A Marshall

    2004-09-15

    The ATRX protein, associated with X-linked alpha-thalassaemia, mental retardation and developmental abnormalities including genital dysgenesis, has been proposed to function as a global transcriptional regulator within a multi-protein complex. However, an understanding of the composition and mechanics of this machinery has remained elusive. We applied inter-specific comparative analysis to identify conserved elements which may be involved in regulating the conformation of chromatin. As part of this study, we cloned and sequenced the entire translatable coding region (7.4 kb) of the ATRX gene from a model marsupial (tammar wallaby, Macropus eugenii). We identify an ATRX ancestral core, conserved between plants, fish and mammals, comprising the cysteine-rich and SWI2/SNF2 helicase-like regions and protein interaction domains. Our data are consistent with the model of the cysteine-rich region as a DNA-binding zinc finger adjacent to a protein-binding (plant homeodomain-like) domain. Alignment of vertebrate ATRX sequences highlights other conserved elements, including a negatively charged mammalian sequence which we propose to be involved in binding of positively charged histone tails.

  20. Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

    PubMed

    Merino, Felipe; Bouvier, Benjamin; Cojocaru, Vlad

    2015-06-01

    Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

  1. Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery

    PubMed Central

    Merino, Felipe; Bouvier, Benjamin; Cojocaru, Vlad

    2015-01-01

    Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions. PMID:26067358

  2. Protein folding and unfolding in microseconds to nanoseconds by experiment and simulation

    PubMed Central

    Mayor, Ugo; Johnson, Christopher M.; Daggett, Valerie; Fersht, Alan R.

    2000-01-01

    The Engrailed Homeodomain protein has the highest refolding and unfolding rate constants directly observed to date. Temperature jump relaxation measurements gave a refolding rate constant of 37,500 s−1 in water at 25°C, rising to 51,000 s−1 around 42°C. The unfolding rate constant was 1,100 s−1 in water at 25°C and 205,000 s−1 at 63°C. The unfolding half-life is extrapolated to be ≈7.5 ns at 100°C, which allows real-time molecular dynamics unfolding simulations to be tested on this system at a realistic temperature. Preliminary simulations did indeed conform to unfolding on this time scale. Further, similar transition states were observed in simulations at 100°C and 225°C, suggesting that high-temperature simulations provide results applicable to lower temperatures. PMID:11087839

  3. Prediction and validation of protein-protein interactors from genome-wide DNA-binding data using a knowledge-based machine-learning approach.

    PubMed

    Waardenberg, Ashley J; Homan, Bernou; Mohamed, Stephanie; Harvey, Richard P; Bouveret, Romaric

    2016-09-01

    The ability to accurately predict the DNA targets and interacting cofactors of transcriptional regulators from genome-wide data can significantly advance our understanding of gene regulatory networks. NKX2-5 is a homeodomain transcription factor that sits high in the cardiac gene regulatory network and is essential for normal heart development. We previously identified genomic targets for NKX2-5 in mouse HL-1 atrial cardiomyocytes using DNA-adenine methyltransferase identification (DamID). Here, we apply machine learning algorithms and propose a knowledge-based feature selection method for predicting NKX2-5 protein : protein interactions based on motif grammar in genome-wide DNA-binding data. We assessed model performance using leave-one-out cross-validation and a completely independent DamID experiment performed with replicates. In addition to identifying previously described NKX2-5-interacting proteins, including GATA, HAND and TBX family members, a number of novel interactors were identified, with direct protein : protein interactions between NKX2-5 and retinoid X receptor (RXR), paired-related homeobox (PRRX) and Ikaros zinc fingers (IKZF) validated using the yeast two-hybrid assay. We also found that the interaction of RXRα with NKX2-5 mutations found in congenital heart disease (Q187H, R189G and R190H) was altered. These findings highlight an intuitive approach to accessing protein-protein interaction information of transcription factors in DNA-binding experiments. © 2016 The Authors.

  4. Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis.

    PubMed

    Zhang, Liguo; Zhang, Xiaofei; Ju, Hanxun; Chen, Jingui; Wang, Shucai; Wang, Hemeng; Zhao, Yuanling; Chang, Ying

    2016-02-12

    Three-Amino-acid-Loop-Extension(TALE) homeodomain transcription factor BLH3 regulates timing of transition from vegetative to reproductive phase. Previous preliminary results obtained using large-scale yeast two-hybrids indicate that BLH3 protein possibly interact with Ovate Family Proteins(OFPs) transcription co-regulators. Nevertheless, it is uncertain whether OFP1-BLH3 complex is involved in regulation of timing of transition from vegetative to reproductive phase in Arabidopsis. The interaction between BLH3 and OFP1 was re-tested and verified by a yeast two-hybrid system. We found that the BLH3-OFP1 interaction was mainly mediated through the BLH3 homeodomain. Meanwhile, this interaction was further confirmed by bimolecular fluorescence complementation (BiFC) in vivo. Further, by establishing protoplast transient expression, we discovered that BLH3 acts as a transcriptional activator, whereas OFP1 functioned as a repressor. The interactions between OFP1 and BLH3 can reduce BLH3 transcriptional activity. The ofp1 mutant lines and blh3 mutant lines, OFP1 overexpress lines and BLH3 overexpress lines can both influence timing of transition from vegetative to reproductive phase. Furthermore, 35s:OFP1/blh3 plants exhibited flowering and leaf quantity similar to that of the wild-type controls. 35s:BLH3/ofp1 plants flowered earlier and had less leaves than wild-type controls, indicating that OFP1 protein might depend partially on BLH3 in its function to regulate the timing of transition from vegetative to reproductive phase. These results support our assumption that, by interacting with OFP1, BLH3 forms a functional protein complex that controls timing of progression from vegetative to reproductive phase, and OFP1 might negatively regulate BLH3 or the BLH-KNOX complex, an important interaction for sustaining the normal transition from vegetative to reproductive phase. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Multimolecule test-tube simulations of protein unfolding and aggregation

    PubMed Central

    McCully, Michelle E.; Beck, David A. C.; Daggett, Valerie

    2012-01-01

    Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level. PMID:23091038

  6. The C. elegans protein CEH-30 protects male-specific neurons from apoptosis independently of the Bcl-2 homolog CED-9.

    PubMed

    Schwartz, Hillel T; Horvitz, H Robert

    2007-12-01

    The developmental control of apoptosis is fundamental and important. We report that the Caenorhabditis elegans Bar homeodomain transcription factor CEH-30 is required for the sexually dimorphic survival of the male-specific CEM (cephalic male) sensory neurons; the homologous cells of hermaphrodites undergo programmed cell death. We propose that the cell-type-specific anti-apoptotic gene ceh-30 is transcriptionally repressed by the TRA-1 transcription factor, the terminal regulator of sexual identity in C. elegans, to cause hermaphrodite-specific CEM death. The established mechanism for the regulation of specific programmed cell deaths in C. elegans is the transcriptional control of the BH3-only gene egl-1, which inhibits the Bcl-2 homolog ced-9; similarly, most regulation of vertebrate apoptosis involves the Bcl-2 superfamily. In contrast, ceh-30 acts within the CEM neurons to promote their survival independently of both egl-1 and ced-9. Mammalian ceh-30 homologs can substitute for ceh-30 in C. elegans. Mice lacking the ceh-30 homolog Barhl1 show a progressive loss of sensory neurons and increased sensory-neuron cell death. Based on these observations, we suggest that the function of Bar homeodomain proteins as cell-type-specific inhibitors of apoptosis is evolutionarily conserved.

  7. Unconventional interactions between water and heterocyclic nitrogens in protein structures.

    PubMed

    Stollar, Elliott J; Gelpí, Jose Luis; Velankar, Sameer; Golovin, Adel; Orozco, Modesto; Luisi, Ben F

    2004-10-01

    We report an unusual interaction in which a water molecule approaches the heterocyclic nitrogen of tryptophan and histidine along an axis that is roughly perpendicular to the aromatic plane of the side chain. The interaction is distinct from the well-known conventional aromatic hydrogen-bond, and it occurs at roughly the same frequency in protein structures. Calculations indicate that the water-indole interaction is favorable energetically, and we find several cases in which such contacts are conserved among structural orthologs. The indole-water interaction links side chains and peptide backbone in turn regions, connects the side chains in beta-sheets, and bridges secondary elements from different domains. We suggest that the water-indole interaction can be indirectly responsible for the quenching of tryptophan fluorescence that is observed in the folding of homeodomains and, possibly, many other proteins. We also observe a similar interaction between water and the imidazole nitrogens of the histidine side chain. Taken together, these observations suggest that the unconventional water-indole and water-imidazole interactions provide a small but favorable contribution to protein structures.

  8. Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

    PubMed Central

    Xu, Zhixiong; Meng, Xianzhang; Cai, Ying; Liang, Hong; Nagarajan, Lalitha; Brandt, Stephen J.

    2007-01-01

    The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins. PMID:17437998

  9. Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context.

    PubMed

    Viganò, M A; Di Rocco, G; Zappavigna, V; Mavilio, F

    1998-11-01

    Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

  10. Control of stem cell homeostasis via interlocking microRNA and microProtein feedback loops.

    PubMed

    Brandt, Ronny; Xie, Yakun; Musielak, Thomas; Graeff, Moritz; Stierhof, York-Dieter; Huang, Hai; Liu, Chun-Ming; Wenkel, Stephan

    2013-01-01

    Stem cells in the shoot apex of plants produce cells required for the formation of new leaves. Adult leaves are composed of multiple tissue layers arranged along the dorso-ventral (adaxial/abaxial) axis. Class III homeodomain leucine zipper (HD-ZIPIII) transcription factors play an important role in the set-up of leaf polarity in plants. Loss of HD-ZIPIII function results in strongly misshapen leaves and in severe cases fosters the consumption of the apical stem cells, thus causing a growth arrest in mutant plants. HD-ZIPIII mRNA is under tight control by microRNAs 165/166. In addition to the microRNA-action a second layer of regulation is established by LITTLE ZIPPER (ZPR)-type microProteins, which can interact with HD-ZIPIII proteins, forming attenuated protein complexes. Here we show that REVOLUTA (REV, a member of the HD-ZIPIII family) directly regulates the expression of ARGONAUTE10 (AGO10), ZPR1 and ZPR3. Because AGO10 was shown to dampen microRNA165/6 function, REV establishes a positive feedback loop on its own activity. Since ZPR-type microProteins are known to reduce HD-ZIPIII protein activity, REV concomitantly establishes a negative feedback loop. We propose that the interconnection of these microRNA/microProtein feedback loops regulates polarity set-up and stem cell activity in plants.

  11. Universality and diversity of folding mechanics for three-helix bundle proteins.

    PubMed

    Yang, Jae Shick; Wallin, Stefan; Shakhnovich, Eugene I

    2008-01-22

    In this study we evaluate, at full atomic detail, the folding processes of two small helical proteins, the B domain of protein A and the Villin headpiece. Folding kinetics are studied by performing a large number of ab initio Monte Carlo folding simulations using a single transferable all-atom potential. Using these trajectories, we examine the relaxation behavior, secondary structure formation, and transition-state ensembles (TSEs) of the two proteins and compare our results with experimental data and previous computational studies. To obtain a detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Moreover, rigorous p(fold) analysis is used to obtain representative samples of the TSEs and a good quantitative agreement between experimental and simulated Phi values is obtained for protein A. Phi values for Villin also are obtained and left as predictions to be tested by future experiments. Our analysis shows that the two-helix hairpin is a common partially stable structural motif that gets formed before entering the TSE in the studied proteins. These results together with our earlier study of Engrailed Homeodomain and recent experimental studies provide a comprehensive, atomic-level picture of folding mechanics of three-helix bundle proteins.

  12. [Protein transduction, from technology to physiology].

    PubMed

    Prochiantz, Alain

    2006-01-01

    In the early 90s, we found that the DNA-binding domain (homeodomain) of Antennapedia, a homeoprotein transcription factor, was internalized by live cells gaining access to their cytoplasm and nuclei. It was soon revealed that internalization is due to the third helix of the homeodomain, composed of sixteen amino acids. This short peptide baptized Penetratin is the first of a large series of transduction peptides widely used for the internalization of all sorts of cargoes in vitro and in vivo. Although transduction peptides are being developed with the latter practical goal, the most intriguing outcome of our initial observation is that full-length homeoproteins are transferred between cells and have non-cell autonomous transcriptional and translational activities. This new signaling mechanism requires that homeoproteins be internalized and secreted. Secretion is Golgi independent and requires a small sequence also present in the homeodomain but distinct from the Penetratin sequence. The consequences of this novel signaling mechanism are briefly discussed.

  13. Paired related homeobox protein-like 1 (Prrxl1) controls its own expression by a transcriptional autorepression mechanism.

    PubMed

    Monteiro, César B; Costa, Mariana F; Reguenga, Carlos; Lima, Deolinda; Castro, Diogo S; Monteiro, Filipe A

    2014-09-17

    The homeodomain factor paired related homeobox protein-like 1 (Prrxl1) is crucial for proper assembly of dorsal root ganglia (DRG)-dorsal spinal cord (SC) pain-sensing circuit. By performing chromatin immunoprecipitation with either embryonic DRG or dorsal SC, we identified two evolutionarily conserved regions (i.e. proximal promoter and intron 4) of Prrxl1 locus that show tissue-specific binding of Prrxl1. Transcriptional assays confirm the identified regions can mediate repression by Prrxl1, while gain-of-function studies in Prrxl1 expressing ND7/23 cells indicate Prrxl1 can down-regulate its own expression. Altogether, our results suggest that Prrxl1 uses distinct regulatory regions to repress its own expression in DRG and dorsal SC.

  14. Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

    PubMed Central

    Morcillo, Patrick; Rosen, Christina; Baylies, Mary K.; Dorsett, Dale

    1997-01-01

    The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer–promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer–promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair–rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer–promoter interactions. PMID:9334334

  15. Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis

    SciTech Connect

    Russell, Michael; Berardi, Philip; Gong Wei; Riabowol, Karl . E-mail: karl@ucalgary.ca

    2006-04-15

    The INhibitor of Growth (ING) family of plant homeodomain (PHD) proteins induce apoptosis and regulate gene expression through stress-inducible binding of phospholipids with subsequent nuclear and nucleolar localization. Relocalization occurs concomitantly with interaction with a subset of nuclear proteins, including PCNA, p53 and several regulators of acetylation such as the p300/CBP and PCAF histone acetyltransferases (HATs), as well as the histone deacetylases HDAC1 and hSir2. These interactions alter the localized state of chromatin compaction, subsequently affecting the expression of subsets of genes, including those associated with the stress response (Hsp70), apoptosis (Bax, MDM2) and cell cycle regulation (p21{sup WAF1}, cyclin B) in a cell- and tissue-specific manner. The expression levels and subcellular localization of ING proteins are altered in a significant number of human cancer types, while the expression of ING isoforms changes during cellular aging, suggesting that ING proteins may play a role in linking cellular transformation and replicative senescence. The variety of functions attributed to ING proteins suggest that this tumor suppressor serves to link the disparate processes of cell cycle regulation, cell suicide and cellular aging through epigenetic regulation of gene expression. This review examines recent findings in the ING field with a focus on the functions of protein-protein interactions involving ING family members and the mechanisms by which these interactions facilitate the various roles that ING proteins play in tumorigenesis, apoptosis and senescence.

  16. Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox proteins.

    PubMed

    Ferretti, E; Marshall, H; Pöpperl, H; Maconochie, M; Krumlauf, R; Blasi, F

    2000-01-01

    Direct auto- and cross-regulatory interactions between Hox genes serve to establish and maintain segmentally restricted patterns in the developing hindbrain. Rhombomere r4-specific expression of both Hoxb1 and Hoxb2 depends upon bipartite cis Hox response elements for the group 1 paralogous proteins, Hoxal and Hoxbl. The DNA-binding ability and selectivity of these proteins depend upon the formation of specific heterodimeric complexes with members of the PBC homeodomain protein family (Pbx genes). The r4 enhancers from Hoxb1 and Hoxb2 have the same activity, but differ with respect to the number and organisation of bipartite Pbx/Hox (PH) sites required, suggesting the intervention of other components/sequences. We report here that another family of homeodomain proteins (TALE, Three-Amino acids-Loop-Extension: Prep1, Meis, HTH), capable of dimerizing with Pbx/EXD, is involved in the mechanisms of r4-restricted expression. We show that: (1) the r4-specific Hoxb1 and Hoxb2 enhancers are complex elements containing separate PH and Prep/Meis (PM) sites; (2) the PM site of the Hoxb2, but not Hoxb1, enhancer is essential in vivo for r4 expression and also influences other sites of expression; (3) both PM and PH sites are required for in vitro binding of Prepl-Pbx and formation and binding of a ternary Hoxbl-Pbxla (or 1b)-Prepl complex. (4) A similar ternary association forms in nuclear extracts from embryonal P19 cells, but only upon retinoic acid induction. This requires synthesis of Hoxbl and also contains Pbx with either Prepl or Meisl. Together these findings highlight the fact that PM sites are found in close proximity to bipartite PH motifs in several Hox responsive elements shown to be important in vivo and that such sites play an essential role in potentiating regulatory activity in combination with the PH motifs.

  17. Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration1[C][W

    PubMed Central

    Guerra, Davide; Mastrangelo, Anna Maria; Lopez-Torrejon, Gema; Marzin, Stephan; Schweizer, Patrick; Stanca, Antonio Michele; del Pozo, Juan Carlos; Cattivelli, Luigi; Mazzucotelli, Elisabetta

    2012-01-01

    Plants exploit ubiquitination to modulate the proteome with the final aim to ensure environmental adaptation and developmental plasticity. Ubiquitination targets are specifically driven to degradation through the action of E3 ubiquitin ligases. Genetic analyses have indicated wide functions of ubiquitination in plant life; nevertheless, despite the large number of predicted E3s, only a few of them have been characterized so far, and only a few ubiquitination targets are known. In this work, we characterized durum wheat (Triticum durum) RING Finger1 (TdRF1) as a durum wheat nuclear ubiquitin ligase. Moreover, its barley (Hordeum vulgare) homolog was shown to protect cells from dehydration stress. A protein network interacting with TdRF1 has been defined. The transcription factor WHEAT BEL1-TYPE HOMEODOMAIN1 (WBLH1) was degraded in a TdRF1-dependent manner through the 26S proteasome in vivo, the mitogen-activated protein kinase TdWNK5 [for Triticum durum WITH NO LYSINE (K)5] was able to phosphorylate TdRF1 in vitro, and the RING-finger protein WHEAT VIVIPAROUS-INTERACTING PROTEIN2 (WVIP2) was shown to have a strong E3 ligase activity. The genes coding for the TdRF1 interactors were all responsive to cold and/or dehydration stress, and a negative regulative function in dehydration tolerance was observed for the barley homolog of WVIP2. A role in the control of plant development was previously known, or predictable based on homology, for wheat BEL1-type homeodomain1(WBLH1). Thus, TdRF1 E3 ligase might act regulating the response to abiotic stress and remodeling plant development in response to environmental constraints. PMID:22167118

  18. The Phosphorylation of PDX-1 by Protein Kinase CK2 Is Crucial for Its Stability

    PubMed Central

    Klein, Sabrina; Meng, Rui; Montenarh, Mathias; Götz, Claudia

    2016-01-01

    The homeodomain protein PDX-1 is a critical regulator of pancreatic development and insulin production in pancreatic β-cells. We have recently shown that PDX-1 is a substrate of protein kinase CK2; a multifunctional protein kinase which is implicated in the regulation of various cellular aspects, such as differentiation, proliferation, and survival. The CK2 phosphorylation site of PDX-1 is located within the binding region of the E3 ubiquitin ligase adaptor protein PCIF1. To study the interaction between PDX-1 and PCIF1 we used immunofluorescence analysis, co-immunoprecipitation, GST-pull-down studies, and proximity ligation assay (PLA). For the analysis of the stability of PDX-1 we performed a cycloheximide chase. We used PDX-1 in its wild-type form as well as phosphomutants of the CK2 phosphorylation site. In pancreatic β-cells PDX-1 binds to PCIF1. The phosphorylation of PDX-1 by CK2 increases the ratio of PCIF1 bound to PDX-1. The stability of PDX-1 is extended in the absence of CK2 phosphorylation. Our results identified protein kinase CK2 as new important modulator of the stability of PDX-1. PMID:28036027

  19. In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

    PubMed Central

    Urbanus, Susan L; de Folter, Stefan; Shchennikova, Anna V; Kaufmann, Kerstin; Immink, Richard GH; Angenent, Gerco C

    2009-01-01

    Background MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during

  20. A STE12 homologue of the homothallic ascomycete Sordaria macrospora interacts with the MADS box protein MCM1 and is required for ascosporogenesis.

    PubMed

    Nolting, Nicole; Pöggeler, Stefanie

    2006-11-01

    The MADS box protein MCM1 controls diverse developmental processes and is essential for fruiting body formation in the homothallic ascomycete Sordaria macrospora. MADS box proteins derive their regulatory specificity from a wide range of different protein interactions. We have recently shown that the S. macrospora MCM1 is able to interact with the alpha-domain mating-type protein SMTA-1. To further evaluate the functional roles of MCM1, we used the yeast two-hybrid approach to identify MCM1-interacting proteins. From this screen, we isolated a protein with a putative N-terminal homeodomain and C-terminal C2/H2-Zn2+ finger domains. The protein is a member of the highly conserved fungal STE12 transcription factor family of proteins and was therefore termed STE12. Furthermore, we demonstrate by means of two-hybrid and far western analysis that in addition to MCM1, the S. macrospora STE12 protein is able to interact with the mating-type protein SMTA-1. Unlike the situation in the closely related heterothallic ascomycete Neurospora crassa, deletion (Delta) of the ste12 gene in S. macrospora neither affects vegetative growth nor fruiting body formation. However, ascus and ascospore development are highly impaired by the Deltaste12 mutation. Our data provide another example of the functional divergence within the fungal STE12 transcription factor family.

  1. Ultrabithorax protein is necessary but not sufficient for full activation of decapentaplegic expression in the visceral mesoderm.

    PubMed Central

    Sun, B; Hursh, D A; Jackson, D; Beachy, P A

    1995-01-01

    To elucidate the mechanisms by which homeotic selector (HOM) genes specify the unique features of Drosophila segments, we have analyzed the regulation of decapentaplegic (dpp), a transforming growth factor (TGF)-beta superfamily member, and have found that the Ultrabithorax (Ubx) HOM protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle (exd). The exd protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require Ubx protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element. Thus, even in an apparently simple case where visceral mesoderm expression of the dpp target gene mirrors that of the Ubx HOM protein, full activation by Ubx protein requires at least one additional factor. In addition, a distinct regulatory mode not directly involving Ubx protein also appears to contribute to PS7-specific expression. Images PMID:7859741

  2. Conservation and diversification of Msx protein in metazoan evolution.

    PubMed

    Takahashi, Hirokazu; Kamiya, Akiko; Ishiguro, Akira; Suzuki, Atsushi C; Saitou, Naruya; Toyoda, Atsushi; Aruga, Jun

    2008-01-01

    Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family

  3. Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

    PubMed Central

    Sauvegarde, Caroline; Paul, Delphine; Bridoux, Laure; Jouneau, Alice; Degrelle, Séverine; Hue, Isabelle; Rezsohazy, René; Donnay, Isabelle

    2016-01-01

    Background We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. Results The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. Conclusions Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals. PMID:27798681

  4. PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein

    PubMed Central

    Todd, Matthew A.M.; Ivanochko, Danton; Picketts, David J.

    2015-01-01

    The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6) gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson–Forssman–Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis. PMID:26103525

  5. The Chromatin-Associated Phf12 Protein Maintains Nucleolar Integrity and Prevents Premature Cellular Senescence

    PubMed Central

    Graveline, Richard; Marcinkiewicz, Katarzyna; Choi, Seyun; Paquet, Marilène; Wurst, Wolfgang; Floss, Thomas

    2016-01-01

    ABSTRACT Pf1, also known as Phf12 (plant homeodomain [PHD] zinc finger protein 12), is a member of the PHD zinc finger family of proteins. Pf1 associates with a chromatin-interacting protein complex comprised of MRG15, Sin3B, and histone deacetylase 1 (HDAC1) that functions as a transcriptional modulator. The biological function of Pf1 remains largely elusive. We undertook the generation of Pf1 knockout mice to elucidate its physiological role. We demonstrate that Pf1 is required for mid- to late gestation viability. Pf1 inactivation impairs the proliferative potential of mouse embryonic fibroblasts (MEFs) and is associated with a significant decrease in bromodeoxyuridine incorporation; an increase in senescence-associated β-galactosidase (SA-β-Gal) activity, a marker of cellular senescence; and elevated levels of phosphorylated H2AX (γ-H2A.X), a marker associated with DNA double-strand breaks. Analysis of transcripts differentially expressed in wild-type and Pf1-deficient cells revealed the impact of Pf1 in multiple regulatory arms of the ribosome biogenesis pathways. Strikingly, assessment of the morphology of the nucleoli exposed an abnormal nucleolar structure in Pf1-deficient cells. Finally, proteomic analysis of the Pf1-interacting complexes highlighted proteins involved in ribosome biogenesis. Taken together, our data reveal an unsuspected function for the Pf1-associated chromatin complex in the ribosomal biogenesis and senescence pathways. PMID:27956701

  6. Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice

    PubMed Central

    Claiborn, Kathryn C.; Sachdeva, Mira M.; Cannon, Corey E.; Groff, David N.; Singer, Jeffrey D.; Stoffers, Doris A.

    2010-01-01

    The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the β cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus–interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse β cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1+/– mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and β cell function and normalized β cell mass in Pdx1+/– mice by modulating β cell survival. These findings indicate that in adult mouse β cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of β cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis. PMID:20811152

  7. PARP1 regulates the protein stability and proapoptotic function of HIPK2

    PubMed Central

    Choi, Jong-Ryoul; Shin, Ki Soon; Choi, Cheol Yong; Kang, Shin Jung

    2016-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in DNA damage response and development. In the present study, we propose that the protein stability and proapoptotic function of HIPK2 are regulated by poly(ADP-ribose) polymerase 1 (PARP1). We present evidence indicating that PARP1 promotes the proteasomal degradation of HIPK2. The tryptophan-glycine-arginine (WGR) domain of PARP1 was necessary and sufficient for the promotion of HIPK2 degradation independently of the PARP1 enzymatic activity. The WGR domain mediated the interaction between HIPK2 and C-terminus of HSP70-interacting protein (CHIP) via HSP70. We found that CHIP can function as a ubiquitin ligase for HIPK2. The interaction between PAPR1 and HIPK2 was weakened following DNA damage. Importantly, PARP1 reduced the HIPK2-mediated p53 phosphorylation, proapoptotic transcriptional activity and cell death. These results suggest that PARP1 can modulate the tumor-suppressing function of HIPK2 by regulating the protein stability of HIPK2. PMID:27787517

  8. Long-term high animal protein diet reduces body weight gain and insulin secretion in diet-induced obese rats.

    PubMed

    Chen, Haiyan; Wang, Yiling; Ma, Lichuan; Zhao, Jiajun; Li, Yinyin; Li, Minglong

    2012-10-01

    The effects of a high protein diet on insulin secretion and glucose metabolism have been quite controversial. The aim of this study was to evaluate the effects of long-term isocaloric high animal protein intake on insulin secretion in diet-induced obese rats. After the experimental period (24 weeks), the high-fat diet-induced obese rats that were fed isocaloric high-protein diets (HP) had lower body weight gain (P < 0.01) and lower visceral fat (P < 0.05) than normal protein (NP) rats. Fasting plasma glucagon-like peptide-1 (GLP-1) was also reduced significantly (P < 0.05), as well as serum insulin levels at 5 min and 10 min by intravenous insulin releasing test. In addition, insulin mRNA and pancreatic duodenal homeodomain-1 (PDX-1), GLP-1 protein expression were both markedly lower in HP rats (P < 0.05), while PDX-1 mRNA in HP rats had no difference from NP rats. These results suggest that long-term isocaloric high animal protein intake reduces the acute insulin response in obese rats and the decrease of insulin is associated with both reduced weight gain and inhibition of PDX-1 expression. GLP-1 might be a negative feedback for the balance of energy metabolism secondary to changes of body weight and visceral fat. Copyright © 2012 Society of Chemical Industry.

  9. Structural and Functional Insights into the Human Börjeson-Forssman-Lehmann Syndrome-associated Protein PHF6*

    PubMed Central

    Liu, Zhonghua; Li, Fudong; Ruan, Ke; Zhang, Jiahai; Mei, Yide; Wu, Jihui; Shi, Yunyu

    2014-01-01

    The plant homeodomain finger 6 (PHF6) was originally identified as the gene mutated in the X-linked mental retardation disorder Börjeson-Forssman-Lehmann syndrome. Mutations in the PHF6 gene have also been associated with T-cell acute lymphoblastic leukemia and acute myeloid leukemia. Approximately half of the disease-associated mutations are distributed in the second conserved extended plant homeodomain (ePHD2) of PHF6, indicating the functional importance of the ePHD2 domain. Here, we report the high resolution crystal structure of the ePHD2 domain of PHF6, which contains an N-terminal pre-PHD (C2HC zinc finger), a long linker, and an atypical PHD finger. PHF6-ePHD2 appears to fold as a novel integrated structural module. Structural analysis of PHF6-ePHD2 reveals pathological implication of PHF6 gene mutations in Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, and acute myeloid leukemia. The binding experiments show that PHF6-ePHD2 can bind dsDNA but not histones. We also demonstrate PHF6 protein directly interacts with the nucleosome remodeling and deacetylation complex component RBBP4. Via this interaction, PHF6 exerts its transcriptional repression activity. Taken together, these data support the hypothesis that PHF6 may function as a transcriptional repressor using its ePHD domains binding to the promoter region of its repressed gene, and this process was regulated by the nucleosome remodeling and deacetylation complex that was recruited to the genomic target site by NoLS region of PHF6. PMID:24554700

  10. Intrinsically disordered regions as affinity tuners in protein-DNA interactions.

    PubMed

    Vuzman, Dana; Levy, Yaakov

    2012-01-01

    Intrinsically disordered regions, terminal tails, and flexible linkers are abundant in DNA-binding proteins and play a crucial role by increasing the affinity and specificity of DNA binding. Disordered tails often undergo a disorder-to-order transition during interactions with DNA and improve both the kinetics and thermodynamics of specific DNA binding. The DNA search by proteins that interact nonspecifically with DNA can be supported by disordered tails as well. The disordered tail may increase the overall protein-DNA interface and thus increase the affinity of the protein to the DNA and its sliding propensity while slowing linear diffusion. The exact effect of the disordered tails on the sliding rate depends on the degree of positive charge clustering, as has been shown for homeodomains and p53 transcription factors. The disordered tails, which may be viewed as DNA recognizing subdomains, can facilitate intersegment transfer events that occur via a "monkey bar" mechanism in which the domains bridge two different DNA fragments simultaneously. The "monkey bar" mechanism can be facilitated by internal disordered linkers in multidomain proteins that mediate the cross-talks between the constituent domains and especially their brachiation dynamics and thus their overall capability to search DNA efficiently. The residue sequence of the disordered tails has unique characteristics that were evolutionarily selected to achieve the optimized function that is unique to each protein. Perturbation of the electrostatic characteristics of the disordered tails by post-translational modifications, such as acetylation and phosphorylation, may affect protein affinity to DNA and therefore can serve to regulate DNA recognition. Modifying the disordered protein tails or the flexibility of the inter-domain linkers of multidomain proteins may affect the cross-talk between the constituent domains so as to facilitate the search kinetics of non-specific DNA sequences and increase affinity to

  11. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

    PubMed

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

  12. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype

    PubMed Central

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis. PMID:27031510

  13. The LIM motif defines a specific zinc-binding protein domain.

    PubMed

    Michelsen, J W; Schmeichel, K L; Beckerle, M C; Winge, D R

    1993-05-15

    The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.

  14. Chromatin-Dependent Repression of the Arabidopsis Floral Integrator Genes Involves Plant Specific PHD-Containing Proteins[C][W

    PubMed Central

    López-González, Leticia; Mouriz, Alfonso; Narro-Diego, Laura; Bustos, Regla; Martínez-Zapater, José Miguel; Jarillo, Jose A.; Piñeiro, Manuel

    2014-01-01

    The interplay among histone modifications modulates the expression of master regulatory genes in development. Chromatin effector proteins bind histone modifications and translate the epigenetic status into gene expression patterns that control development. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific proteins with a plant homeodomain (PHD) motif, SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), function in the chromatin-mediated repression of floral initiation and play independent roles in the control of genes regulating flowering. Previous results showed that repression of the floral integrator FLOWERING LOCUS T (FT) requires EBS. We establish that SHL is necessary to negatively regulate the expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), another floral integrator. SHL and EBS recognize di- and trimethylated histone H3 at lysine 4 and bind regulatory regions of SOC1 and FT, respectively. These PHD proteins maintain an inactive chromatin conformation in SOC1 and FT by preventing high levels of H3 acetylation, bind HISTONE DEACETYLASE6, and play a central role in regulating flowering time. SHL and EBS are widely conserved in plants but are absent in other eukaryotes, suggesting that the regulatory module mediated by these proteins could represent a distinct mechanism for gene expression control in plants. PMID:25281686

  15. Chromatin-dependent repression of the Arabidopsis floral integrator genes involves plant specific PHD-containing proteins.

    PubMed

    López-González, Leticia; Mouriz, Alfonso; Narro-Diego, Laura; Bustos, Regla; Martínez-Zapater, José Miguel; Jarillo, Jose A; Piñeiro, Manuel

    2014-10-01

    The interplay among histone modifications modulates the expression of master regulatory genes in development. Chromatin effector proteins bind histone modifications and translate the epigenetic status into gene expression patterns that control development. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific proteins with a plant homeodomain (PHD) motif, SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), function in the chromatin-mediated repression of floral initiation and play independent roles in the control of genes regulating flowering. Previous results showed that repression of the floral integrator FLOWERING LOCUS T (FT) requires EBS. We establish that SHL is necessary to negatively regulate the expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), another floral integrator. SHL and EBS recognize di- and trimethylated histone H3 at lysine 4 and bind regulatory regions of SOC1 and FT, respectively. These PHD proteins maintain an inactive chromatin conformation in SOC1 and FT by preventing high levels of H3 acetylation, bind HISTONE DEACETYLASE6, and play a central role in regulating flowering time. SHL and EBS are widely conserved in plants but are absent in other eukaryotes, suggesting that the regulatory module mediated by these proteins could represent a distinct mechanism for gene expression control in plants.

  16. AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

    PubMed

    Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

    2015-10-01

    Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein. © 2015 John Wiley & Sons Ltd.

  17. Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor.

    PubMed

    Mitton, Kenneth P; Swain, Prabodh K; Khanna, Hemant; Dowd, Mary; Apel, Ingrid J; Swaroop, Anand

    2003-02-15

    NRL (neural retina leucine zipper) is a basic motif leucine zipper transcription factor of the Maf-subfamily. Multiple phosphorylated isoforms of NRL are detected specifically in rod photoreceptors. NRL regulates the expression of several rod-specific genes, including rhodopsin and cGMP phosphodiesterase beta-subunit, in synergy with other transcription factors (e.g. the homeodomain protein CRX). Missense mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa, whereas the loss of its function leads to rodless retina in Nrl-knockout mice that exhibit enhanced S-cone function. To further elucidate the molecular mechanism(s) underlying NRL-mediated transcriptional regulation, we used yeast two-hybrid screening to isolate NRL-interacting proteins in the retina and report the identification of Flt3-interacting zinc-finger protein, Fiz1. Interaction of Fiz1 and NRL-leucine zipper was validated by GST pulldown assays and co-immunoprecipitation from bovine retinal nuclear extracts. Fiz1 suppressed NRL- but not CRX-mediated transactivation of rhodopsin promoter activity in transiently transfected CV1 cells. The mRNA and the protein for both Fiz1 and its only other known interacting protein Flt3, a receptor tyrosine kinase, are expressed in the retina. Our results indicate potential cross-talk among signaling pathways in the retina and suggest that the function of NRL is modulated by its interaction with specific repressor proteins.

  18. Prediction and validation of protein–protein interactors from genome-wide DNA-binding data using a knowledge-based machine-learning approach

    PubMed Central

    Homan, Bernou; Mohamed, Stephanie; Harvey, Richard P.; Bouveret, Romaric

    2016-01-01

    The ability to accurately predict the DNA targets and interacting cofactors of transcriptional regulators from genome-wide data can significantly advance our understanding of gene regulatory networks. NKX2-5 is a homeodomain transcription factor that sits high in the cardiac gene regulatory network and is essential for normal heart development. We previously identified genomic targets for NKX2-5 in mouse HL-1 atrial cardiomyocytes using DNA-adenine methyltransferase identification (DamID). Here, we apply machine learning algorithms and propose a knowledge-based feature selection method for predicting NKX2-5 protein : protein interactions based on motif grammar in genome-wide DNA-binding data. We assessed model performance using leave-one-out cross-validation and a completely independent DamID experiment performed with replicates. In addition to identifying previously described NKX2-5-interacting proteins, including GATA, HAND and TBX family members, a number of novel interactors were identified, with direct protein : protein interactions between NKX2-5 and retinoid X receptor (RXR), paired-related homeobox (PRRX) and Ikaros zinc fingers (IKZF) validated using the yeast two-hybrid assay. We also found that the interaction of RXRα with NKX2-5 mutations found in congenital heart disease (Q187H, R189G and R190H) was altered. These findings highlight an intuitive approach to accessing protein–protein interaction information of transcription factors in DNA-binding experiments. PMID:27683156

  19. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  20. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  1. Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis

    DOE PAGES

    Zhang, Liguo; Zhang, Xiaofei; Ju, Hanxun; ...

    2016-01-23

    We study the Three-Amino-acid-Loop-Extension(TALE) homeodomain transcription factor BLH3 that regulates timing of transition from vegetative to reproductive phase. Previous preliminary results obtained using large-scale yeast two-hybrids indicate that BLH3 protein possibly interact with Ovate Family Proteins(OFPs) transcription co-regulators. Nevertheless, it is uncertain whether OFP1–BLH3 complex is involved in regulation of timing of transition from vegetative to reproductive phase in Arabidopsis. The interaction between BLH3 and OFP1 was re-tested and verified by a yeast two-hybrid system. We found that the BLH3–OFP1 interaction was mainly mediated through the BLH3 homeodomain. Meanwhile, this interaction was further confirmed by bimolecular fluorescence complementation (BiFC) inmore » vivo. In addition, by establishing protoplast transient expression, we discovered that BLH3 acts as a transcriptional activator, whereas OFP1 functioned as a repressor. The interactions between OFP1 and BLH3 can reduce BLH3 transcriptional activity. The ofp1 mutant lines and blh3 mutant lines, OFP1 overexpress lines and BLH3 overexpress lines can both influence timing of transition from vegetative to reproductive phase. Furthermore, 35s:OFP1/blh3 plants exhibited flowering and leaf quantity similar to that of the wild-type controls. 35s:BLH3/ofp1 plants flowered earlier and had less leaves than wild-type controls, indicating that OFP1 protein might depend partially on BLH3 in its function to regulate the timing of transition from vegetative to reproductive phase. In conclusion, these results support our assumption that, by interacting with OFP1, BLH3 forms a functional protein complex that controls timing of progression from vegetative to reproductive phase, and OFP1 might negatively regulate BLH3 or the BLH-KNOX complex, an important interaction for sustaining the normal transition from vegetative to reproductive phase.« less

  2. Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis

    SciTech Connect

    Zhang, Liguo; Zhang, Xiaofei; Ju, Hanxun; Chen, Jingui; Wang, Shucai; Wang, Hemeng; Zhao, Yuanling; Chang, Ying

    2016-01-23

    We study the Three-Amino-acid-Loop-Extension(TALE) homeodomain transcription factor BLH3 that regulates timing of transition from vegetative to reproductive phase. Previous preliminary results obtained using large-scale yeast two-hybrids indicate that BLH3 protein possibly interact with Ovate Family Proteins(OFPs) transcription co-regulators. Nevertheless, it is uncertain whether OFP1–BLH3 complex is involved in regulation of timing of transition from vegetative to reproductive phase in Arabidopsis. The interaction between BLH3 and OFP1 was re-tested and verified by a yeast two-hybrid system. We found that the BLH3–OFP1 interaction was mainly mediated through the BLH3 homeodomain. Meanwhile, this interaction was further confirmed by bimolecular fluorescence complementation (BiFC) in vivo. In addition, by establishing protoplast transient expression, we discovered that BLH3 acts as a transcriptional activator, whereas OFP1 functioned as a repressor. The interactions between OFP1 and BLH3 can reduce BLH3 transcriptional activity. The ofp1 mutant lines and blh3 mutant lines, OFP1 overexpress lines and BLH3 overexpress lines can both influence timing of transition from vegetative to reproductive phase. Furthermore, 35s:OFP1/blh3 plants exhibited flowering and leaf quantity similar to that of the wild-type controls. 35s:BLH3/ofp1 plants flowered earlier and had less leaves than wild-type controls, indicating that OFP1 protein might depend partially on BLH3 in its function to regulate the timing of transition from vegetative to reproductive phase. In conclusion, these results support our assumption that, by interacting with OFP1, BLH3 forms a functional protein complex that controls timing of progression from vegetative to reproductive phase, and OFP1 might negatively regulate BLH3 or the BLH-KNOX complex, an important interaction for sustaining the normal transition from vegetative to reproductive phase.

  3. The FANTASTIC FOUR proteins influence shoot meristem size in Arabidopsis thaliana

    PubMed Central

    2010-01-01

    Background Throughout their lives plants produce new organs from groups of pluripotent cells called meristems, located at the tips of the shoot and the root. The size of the shoot meristem is tightly controlled by a feedback loop, which involves the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA (CLV) proteins. This regulatory circuit is further fine-tuned by morphogenic signals such as hormones and sugars. Results Here we show that a family of four plant-specific proteins, encoded by the FANTASTIC FOUR (FAF) genes, has the potential to regulate shoot meristem size in Arabidopsis thaliana. FAF2 and FAF4 are expressed in the centre of the shoot meristem, overlapping with the site of WUS expression. Consistent with a regulatory interaction between the FAF gene family and WUS, our experiments indicate that the FAFs can repress WUS, which ultimately leads to an arrest of meristem activity in FAF overexpressing lines. The finding that meristematic expression of FAF2 and FAF4 is under negative control by CLV3 further supports the hypothesis that the FAFs are modulators of the genetic circuit that regulates the meristem. Conclusion This study reports the initial characterization of the Arabidopsis thaliana FAF gene family. Our data indicate that the FAF genes form a plant specific gene family, the members of which have the potential to regulate the size of the shoot meristem by modulating the CLV3-WUS feedback loop. PMID:21176196

  4. Functional analysis of a RING domain ankyrin repeat protein that is highly expressed during flower senescence.

    PubMed

    Xu, Xinjia; Jiang, Cai-Zhong; Donnelly, Linda; Reid, Michael S

    2007-01-01

    A gene encoding a RING zinc finger ankyrin repeat protein (MjXB3), a putative E3 ubiquitin ligase, is highly expressed in petals of senescing four o'clock (Mirabilis jalapa) flowers, increasing >40,000-fold during the onset of visible senescence. The gene has homologues in many other species, and the Petunia homologue is strongly up-regulated in senescing Petunia corollas. Silencing the expression of this gene in Petunia, using virus-induced gene silencing, resulted in a 2 d extension in flower life. In Mirabilis, a 2 kb promoter region, 5' upstream of the MjXB3 gene, was isolated. The promoter sequence included putative binding sites for many DNA-binding proteins, including the bZIP, Myb, homeodomain-leucine zipper (HD-Zip), MADS-box, and WRKY transcription factors. The construct containing a 1 kb promoter region immediately upstream of the MjXB3 gene drove the strongest expression of the beta-glucuronidase (GUS) reporter gene in a transient expression assay. In Petunia, GUS expression under the control of this heterologous promoter fragment was specific to senescing flowers. The Mirabilis promoter GUS construct was tested in other flower species; while GUS activity in carnation petals was high during senescence, no expression was detected in three monocotyledonous flowers--daylily (Hemerocallis 'Stella d'Oro'), daffodil (Narcissus pseudonarcissus 'King Alfred'), and orchid (Dendrobium 'Emma White').

  5. Regulation of spinal interneuron development by the Olig-related protein Bhlhb5 and Notch signaling

    PubMed Central

    Skaggs, Kaia; Martin, Donna M.; Novitch, Bennett G.

    2011-01-01

    The neural circuits that control motor activities depend on the spatially and temporally ordered generation of distinct classes of spinal interneurons. Despite the importance of these interneurons, the mechanisms underlying their genesis are poorly understood. Here, we demonstrate that the Olig-related transcription factor Bhlhb5 (recently renamed Bhlhe22) plays two central roles in this process. Our findings suggest that Bhlhb5 repressor activity acts downstream of retinoid signaling and homeodomain proteins to promote the formation of dI6, V1 and V2 interneuron progenitors and their differentiated progeny. In addition, Bhlhb5 is required to organize the spatially restricted expression of the Notch ligands and Fringe proteins that both elicit the formation of the interneuron populations that arise adjacent to Bhlhb5+ cells and influence the global pattern of neuronal differentiation. Through these actions, Bhlhb5 helps transform the spatial information established by morphogen signaling into local cell-cell interactions associated with Notch signaling that control the progression of neurogenesis and extend neuronal diversity within the developing spinal cord. PMID:21750031

  6. A framework for the DNA-protein recognition code of the probe helix in transcription factors: the chemical and stereochemical rules.

    PubMed

    Suzuki, M

    1994-04-15

    Understanding the general mechanisms of sequence specific DNA recognition by proteins is a major challenge in structural biology. The existence of a 'DNA recognition code' for proteins, by which certain amino acid residues on a protein surface confer specificity for certain DNA bases, has been the subject of much discussion. However, no simple code has yet been established. The principles of DNA recognition can be described at two levels. The 'chemical' rules describe the partnerships between amino acid side chains and DNA bases making favourable interactions in the major groove of DNA. Here I analyze the occurrence of nucleotide-amino acid contacts in previously determined crystal structures of DNA-protein complexes and find that simple rules pertain. I also describe 'stereochemical' rules for the probe helix type of DNA-binding motif found in certain transcription factors including leucine zipper and homeodomain proteins. These are a consequence of the binding geometry, and specify the amino acid and base positions used for the contacts, and the sizes of residues in the contact interface. The chemical rules can be generalized for any DNA-binding motif, while the stereochemical rules are specific to a particular DNA-binding motif. The recognition code for a particular binding motif can be described by combining the two sets of rules.

  7. A novel PHD-finger protein 14/KIF4A complex overexpressed in lung cancer is involved in cell mitosis regulation and tumorigenesis.

    PubMed

    Zhang, Lin; Huang, Qin; Lou, Jiatao; Zou, Liangjian; Wang, Yiguo; Zhang, Peng; Yang, Guang; Zhang, Junyi; Yu, Lan; Yan, Dai; Zhang, Chenyi; Qiao, Jing; Wang, Shuting; Wang, Sai; Xu, Yongdong; Ji, Hongbin; Chen, Zhengjun; Zhang, Zhe

    2017-02-01

    The plant homeodomain (PHD) finger-containing proteins have been implicated in many human diseases including cancer. In this study, we found that PHF14, a newly identified PHD finger protein, is highly expressed in lung cancer. The high expression level of PHF14 was associated with adenocarcinoma and poor survival in lung cancer patients. Knocking down PHF14 suppressed cancer cell growth and carcinogenesis, while over-expressing PHF14 promoted cell proliferation. During cell division, PHF14 directly bound to and co-localized with KIF4A (a nuclear motor protein involved in lung carcinogenesis) to form a functional complex. Similarly to the effect of KIF4A depletion, silencing PHF14 in several cell lines caused cell mitotic defects, prolonged M phase, and inhibited cell proliferation. What's more, these two proteins had a synergistic effect on cell proliferation and were significantly co-overexpressed in lung cancer tissues. Our data provide new insights into the biological significance of PHD finger proteins and imply that PHF14 may be a potential biomarker for lung cancer.

  8. The gene encoding the VP16-accessory protein HCF (HCFC1) resides in human Xq28 and is highly expressed in fetal tissues and the adult kidney

    SciTech Connect

    Wilson, A.C.; Herr, W.; Parrish, J.E.; Massa, H.F.

    1995-01-20

    After herpes simplex virus (HSV) infection, the viral regulatory protein VP16 activates transcription of the HSV immediate-early promoters by directing complex formation with two cellular proteins, the POU-homeodomain transcription factor Oct-1 and the host cell factor HCF. The function of HCF in uninfected cells is unknown. Here we show by fluorescence in situ hybridization and somatic cell hybrid analysis that the gene encoding human HCF, HCFC1, maps to the q28 region of the X chromosome. Yeast artificial chromosome and cosmid mapping localizes the HCFC1 gene within 100 kb distal of the renal vasopressin type-2 receptor (V2R) gene and adjacent to the renin-binding protein gene (RENBP). The HCFC1 gene is apparently unique. HCF transcripts and protein are most abundant in fetal and placental tissues and cell lines, suggesting a role in cell proliferation. In adults, HCF protein is abundant in the kidney, but not in the brain, a site of latent HSV infection and where HCF levels may influence progression of HSV infection. 42 refs., 3 figs.

  9. Genetic Dissection of Photoreceptor Subtype Specification by the Drosophila melanogaster Zinc Finger Proteins Elbow and No ocelli

    PubMed Central

    Wernet, Mathias F.; Meier, Kerstin M.; Baumann-Klausener, Franziska; Dorfman, Ruslan; Weihe, Ulrich; Labhart, Thomas; Desplan, Claude

    2014-01-01

    The elbow/no ocelli (elb/noc) complex of Drosophila melanogaster encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. Here we show that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the ‘dorsal rim area’ (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. Our data suggest that Elb/Noc genetically interact with Hth, and we identify two conserved domains crucial for this function. Furthermore, we show that Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. We propose that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease. PMID:24625735

  10. The Clp1 Protein Is Required for Clamp Formation and Pathogenic Development of Ustilago maydis[W

    PubMed Central

    Scherer, Mario; Heimel, Kai; Starke, Verena; Kämper, Jörg

    2006-01-01

    In the phytopathogenic fungus Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bE and bW, encoded by the b-mating-type locus. We have identified a b-dependently induced gene, clampless1 (clp1), that is required for the proliferation of dikaryotic filaments in planta. We show that U. maydis hyphae develop structures functionally equivalent to clamp cells that participate in the distribution of nuclei during cell division. In clp1 mutant strains, dikaryotic filaments penetrate the plant cuticle, but development is stalled before the first mitotic division, and the clamp-like structures are not formed. Although clp1 is immediately activated upon b-induction on the transcriptional level, nuclear-localized Clp1 protein is first observed at the stage of plant penetration prior to the first cell division. Induced expression of clp1 strongly interferes with b-dependent gene regulation and blocks b-dependent filament formation and b-dependent cell cycle arrest. We speculate that the Clp1 protein inhibits the activity of the bE/bW heterodimer to facilitate the cell cycle progression during hyphal growth. PMID:16920779

  11. The ePHD protein SPBP interacts with TopBP1 and together they co-operate to stimulate Ets1-mediated transcription

    PubMed Central

    Sjøttem, Eva; Rekdal, Cecilie; Svineng, Gunbjørg; Johnsen, Sylvia Sagen; Klenow, Helle; Uglehus, Rebecca Dale; Johansen, Terje

    2007-01-01

    SPBP (Stromelysin-1 PDGF responsive element binding protein) is a ubiquitously expressed 220 kDa nuclear protein shown to enhance or repress the transcriptional activity of various transcription factors. A yeast two-hybrid screen, with the extended plant homeodomain (ePHD) of SPBP as bait, identified TopBP1 (topoisomerase II β-binding protein 1) as a candidate interaction partner of SPBP. TopBP1 has eight BRCA1 carboxy-terminal (BRCT) domains and is involved in DNA replication, DNA damage responses and in the regulation of gene expression. The interaction between SPBP and TopBP1 was confirmed in vitro and in vivo, and was found to be mediated by the ePHD domain of SPBP and the BRCT6 domain of TopBP1. Both SPBP and TopBP1 enhanced the transcriptional activity of Ets1 on the c-myc P1P2- and matrix metalloproteinase-3 (MMP3) promoters. Together they displayed a more than additive effect. Both proteins were associated with these promoters. The involvement of TopBP1 was dependent on the serine 1159 phosphorylation site, known to be important for transcriptional activation. Depletion of endogenous SPBP by siRNA treatment reduced MMP3 secretion by 50% in phorbol ester-stimulated human fibroblasts. Taken together, our results show that TopBP1 and SPBP interact physically and functionally to co-operate as co-activators of Ets1. PMID:17913746

  12. NDR proteins

    PubMed Central

    Jones, Alan M

    2010-01-01

    N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

  13. Residue-specific force field based on the protein coil library. RSFF1: modification of OPLS-AA/L.

    PubMed

    Jiang, Fan; Zhou, Chen-Yang; Wu, Yun-Dong

    2014-06-26

    Traditional protein force fields use one set of parameters for most of the 20 amino acids (AAs), allowing transferability of the parameters. However, a significant shortcoming is the difficulty to fit the Ramachandran plots of all AA residues simultaneously, affecting the accuracy of the force field. In this Feature Article, we report a new strategy for protein force field parametrization. Backbone and side-chain conformational distributions of all 20 AA residues obtained from protein coil library were used as the target data. The dihedral angle (torsion) potentials and some local nonbonded (1-4/1-5/1-6) interactions in OPLS-AA/L force field were modified such that the target data can be excellently reproduced by molecular dynamics simulations of dipeptides (blocked AAs) in explicit water, resulting in a new force field with AA-specific parameters, RSFF1. An efficient free energy decomposition approach was developed to separate the corrections on ϕ and ψ from the two-dimensional Ramachandran plots. RSFF1 is shown to reproduce the experimental NMR (3)J-coupling constants of AA dipeptides better than other force fields. It has a good balance between α-helical and β-sheet secondary structures. It can successfully fold a set of α-helix proteins (Trp-cage and Homeodomain) and β-hairpins (Trpzip-2, GB1 hairpin), which cannot be consistently stabilized by other state-of-the-art force fields. Interestingly, the RSFF1 force field systematically overestimates the melting temperature (and the stability of native state) of these peptides/proteins. It has a potential application in the simulation of protein folding and protein structure refinement.

  14. Kinetics of chain motions within a protein-folding intermediate

    PubMed Central

    Neuweiler, Hannes; Banachewicz, Wiktor; Fersht, Alan R.

    2010-01-01

    Small proteins can fold remarkably rapidly, even in μs. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 μs, but the final docking of preformed helix1 in I requires approximately 20 μs. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-μs time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-μs and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-μs phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion. PMID:21135210

  15. Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter.

    PubMed

    Galibert, M D; Yavuzer, U; Dexter, T J; Goding, C R

    1999-09-17

    Previous work has established that the melanocyte-specific tyrosinase-related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two "melanocyte-specific elements" termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocyte-specific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and anti-Pax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanocyte lineage is discussed.

  16. Expression of the G protein gamma T1 subunit during zebrafish development

    PubMed Central

    Chen, Hui; Leung, TinChung; Giger, Kathryn E.; Stauffer, Anna M.; Humbert, Jasper E.; Sinha, Soniya; Horstick, Eric J.; Hansen, Carl A.; Robishaw, Janet D.

    2009-01-01

    Here, we report the identification and expression analysis of the zebrafish G protein gamma T1 subunit gene (gngT1) during development. Similar to its human and mouse homologs, we confirm zebrafish gngT1 is expressed in the developing retina, where its transcription overlaps with the photoreceptor cell-specific marker, rhodopsin (rho). Surprisingly, we also show zebrafish gngT1 is expressed in the dorsal diencephalon, where its transcription overlaps with the pineal specific markers, arylalkylamine N-acetyltransferase-2 (annat-2) and extra-ocular rhodopsin (exorh). Analysis of the proximal promoter sequence of the zebrafish gngT1 gene identifies several conserved binding sites for the cone-rod homeobox/orthodenticle (Crx/Otx) homeodomain family of transcription factors. Using a morpholino antisense approach in zebrafish, we show that targeted knockdown of otx5 potently suppresses gngT1 expression in the pineal gland, whereas knockdown of crx markedly reduces gngT1 expression in the retina. Taken together, these data indicate that pineal- and retinal-specific expression of the gngT1 gene are controlled by different transcription factors and exogenous signals. PMID:17306630

  17. Proteins (image)

    MedlinePlus

    ... is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals. The healthiest proteins are the leanest. This means ...

  18. Dietary Proteins

    MedlinePlus

    ... and maintain bones, muscles and skin. We get proteins in our diet from meat, dairy products, nuts, and certain grains ... level of physical activity. Most Americans eat enough protein in their diet.

  19. Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation.

    PubMed

    Nasedkin, Alexandr; Marcellini, Moreno; Religa, Tomasz L; Freund, Stefan M; Menzel, Andreas; Fersht, Alan R; Jemth, Per; van der Spoel, David; Davidsson, Jan

    2015-01-01

    The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.

  20. Chromatin condensation and recruitment of PHD finger proteins to histone H3K4me3 are mutually exclusive

    PubMed Central

    Gatchalian, Jovylyn; Gallardo, Carmen Mora; Shinsky, Stephen A.; Ospina, Ruben Rosas; Liendo, Andrea Mansilla; Krajewski, Krzysztof; Klein, Brianna J.; Andrews, Forest H.; Strahl, Brian D.; M. van Wely, Karel H.; Kutateladze, Tatiana G.

    2016-01-01

    Histone post-translational modifications, and specific combinations they create, mediate a wide range of nuclear events. However, the mechanistic bases for recognition of these combinations have not been elucidated. Here, we characterize crosstalk between H3T3 and H3T6 phosphorylation, occurring in mitosis, and H3K4me3, a mark associated with active transcription. We detail the molecular mechanisms by which H3T3ph/K4me3/T6ph switches mediate activities of H3K4me3-binding proteins, including those containing plant homeodomain (PHD) and double Tudor reader domains. Our results derived from nuclear magnetic resonance chemical shift perturbation analysis, orthogonal binding assays and cell fluorescence microscopy studies reveal a strong anti-correlation between histone H3T3/T6 phosphorylation and retention of PHD finger proteins in chromatin during mitosis. Together, our findings uncover the mechanistic rules of chromatin engagement for H3K4me3-specific readers during cell division. PMID:27016734

  1. Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

    PubMed Central

    Nagawa, Fumikiyo; Ishiguro, Kei-ichiro; Tsuboi, Akio; Yoshida, Tomoyuki; Ishikawa, Akiko; Takemori, Toshitada; Otsuka, Anthony J.; Sakano, Hitoshi

    1998-01-01

    We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA. PMID:9418911

  2. Comparative genomics of the Hlx homeobox gene and protein: conservation of structure and expression from fish to mammals.

    PubMed

    Bates, Michael D; Wells, James M; Venkatesh, Byrappa

    2005-06-06

    Hlx is a homeobox transcription factor gene that is expressed in intestinal and hepatic mesenchyme of the developing mouse embryo and is essential for normal intestinal and hepatic development. Because of the morphological and molecular similarities in the development of the digestive system across species, we hypothesized that the Hlx gene and protein sequences and expression patterns would be conserved among vertebrates. Comparison of the Hlx gene orthologues of human, chimpanzee, mouse, rat, pufferfish (Fugu) and zebrafish demonstrates that these six genes share an identical organization with four exons and three introns. Comparison of the inferred Hlx protein sequences from these and three additional species (chick, Spanish ribbed newt and rainbow trout) reveals significant sequence identity, with identical homeodomains. The expression of Hlx in the mesenchyme of developing chick embryos is highly similar to that of mouse. Fugu Hlx is expressed in a tissue-specific manner that is similar though not identical to that of mouse, suggesting a conservation of Hlx function between mammals and birds. The mammalian and fish Hlx genes share a putative 5' upstream enhancer as well as an inverted repeat containing CCAAT boxes on opposite strands that we have previously shown to be important for mouse Hlx gene expression. These results suggest that the function of Hlx and the mechanisms regulating its expression are highly conserved in mammals, birds, amphibians and fish.

  3. Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

    PubMed Central

    Laughon, A; Boulet, A M; Bermingham, J R; Laymon, R A; Scott, M P

    1986-01-01

    The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus. Images PMID:2879223

  4. Dual transcriptional activities of SIX proteins define their roles in normal and ectopic eye development.

    PubMed

    Anderson, Abigail M; Weasner, Bonnie M; Weasner, Brandon P; Kumar, Justin P

    2012-03-01

    The SIX family of homeodomain-containing DNA-binding proteins play crucial roles in both Drosophila and vertebrate retinal specification. In flies, three such family members exist, but only two, Sine oculis (So) and Optix, are expressed and function within the eye. In vertebrates, the homologs of Optix (Six3 and Six6) and probably So (Six1 and Six2) are also required for proper eye formation. Depending upon the individual SIX protein and the specific developmental context, transcription of target genes can either be activated or repressed. These activities are thought to occur through physical interactions with the Eyes absent (Eya) co-activator and the Groucho (Gro) co-repressor, but the relative contribution that each complex makes to overall eye development is not well understood. Here, we attempt to address this issue by investigating the role that each complex plays in the induction of ectopic eyes in Drosophila. We fused the VP16 activation and Engrailed repressor domains to both So and Optix, and attempted to generate ectopic eyes with these chimeric proteins. Surprisingly, we find that So and Optix must initially function as transcriptional repressors to trigger the formation of ectopic eyes. Both factors appear to be required to repress the expression of non-retinal selector genes. We propose that during early phases of eye development, SIX proteins function, in part, to repress the transcription of non-retinal selector genes, thereby allowing induction of the retina to proceed. This model of repression-mediated induction of developmental programs could have implications beyond the eye and might be applicable to other systems.

  5. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  6. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  7. DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning.

    PubMed

    Rodriguez, Kevin; Perales, Mariano; Snipes, Stephen; Yadav, Ram Kishor; Diaz-Mendoza, Mercedes; Reddy, G Venugopala

    2016-10-11

    The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

  8. Therapeutic proteins.

    PubMed

    Dimitrov, Dimiter S

    2012-01-01

    Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major

  9. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

    PubMed Central

    Ansseau, Eugénie; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q.; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  10. Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

    PubMed Central

    Liu, Bigang; Badeaux, Mark D.; Choy, Grace; Chandra, Dhyan; Shen, Irvin; Jeter, Collene R.; Rycaj, Kiera; Lee, Chia-Fang; Person, Maria D.; Liu, Can; Chen, Yueping; Shen, Jianjun; Jung, Sung Yun; Qin, Jun; Tang, Dean G.

    2014-01-01

    Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ∼99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29–80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ∼22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ∼28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells. PMID:24598770

  11. The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.

    PubMed Central

    Fredericks, W J; Galili, N; Mukhopadhyay, S; Rovera, G; Bennicelli, J; Barr, F G; Rauscher, F J

    1995-01-01

    Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes. PMID:7862145

  12. The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene.

    PubMed Central

    Toonen, R F; Gowan, S; Bingle, C D

    1996-01-01

    The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated. Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3' of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP-CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein-DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP-CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-1 bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5' to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Sp1 and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region. PMID:8687389

  13. Cysteine proteinase-1 and cut protein isoform control dendritic innervation of two distinct sensory fields by a single neuron.

    PubMed

    Lyons, Gray R; Andersen, Ryan O; Abdi, Khadar; Song, Won-Seok; Kuo, Chay T

    2014-03-13

    Dendrites often exhibit structural changes in response to local inputs. Although mechanisms that pattern and maintain dendritic arbors are becoming clearer, processes regulating regrowth, during context-dependent plasticity or after injury, remain poorly understood. We found that a class of Drosophila sensory neurons, through complete pruning and regeneration, can elaborate two distinct dendritic trees, innervating independent sensory fields. An expression screen identified Cysteine proteinase-1 (Cp1) as a critical regulator of this process. Unlike known ecdysone effectors, Cp1-mutant ddaC neurons pruned larval dendrites normally but failed to regrow adult dendrites. Cp1 expression was upregulated/concentrated in the nucleus during metamorphosis, controlling production of a truncated Cut homeodomain transcription factor. This truncated Cut, but not the full-length protein, allowed Cp1-mutant ddaC neurons to regenerate higher-order adult dendrites. These results identify a molecular pathway needed for dendrite regrowth after pruning, which allows the same neuron to innervate distinct sensory fields.

  14. Protein tentacles.

    PubMed

    Harrison, Stephen C

    2017-05-27

    Virus structures were among the earliest illustrations of how regulated protein assembly can proceed by folding of polypeptide-chain segments into complementary sites on partner proteins. I draw on Caspar's image of protein "tentacles" and his metaphor of SV40 pentamers as five-legged, aquatic organisms ("pentopuses") to suggest a helpful vocabulary. "Tentacular interactions" among component subunits organize most subcellular molecular machines. Their selective advantages include facile regulation of both assembly and disassembly by modifying enzymes and by folding chaperones. Copyright © 2017. Published by Elsevier Inc.

  15. Nuclear import of the transcription factor SHOOT MERISTEMLESS depends on heterodimerization with BLH proteins expressed in discrete sub-domains of the shoot apical meristem of Arabidopsis thaliana.

    PubMed

    Cole, Melanie; Nolte, Carolin; Werr, Wolfgang

    2006-01-01

    The gene SHOOT MERISTEMLESS (STM) is required for the initiation and the maintenance of the shoot apical meristem (SAM) in Arabidopsis and encodes a MEINOX/three amino acid loop extension (TALE)-HD-type transcription factor. Translational fusions with the green fluorescent protein showed that STM is not nuclear by default. In a yeast two-hybrid screen performed with a meristem-enriched cDNA library, three interacting BLH (Bel1-like homeodomain) transcription factors were identified. According to bimolecular fluorescence complementation, STM is targeted into the nuclear compartment through heterodimerization with BLH partner proteins, which are expressed in distinct SAM domains from the center to the periphery. On a functional level, overexpression experiments in transgenic Arabidopsis plants suggest that individual heterodimers provide distinct contributions. These results contribute to our understanding of the STM transcription factor function in the SAM and also shed new light on the evolution of the TALE-HD super gene family in animal and plant lineages.

  16. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  17. Protein Crystallizability.

    PubMed

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  18. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  19. Using Molecular Dynamics Simulations as an Aid in the Prediction of Domain Swapping of Computationally Designed Protein Variants.

    PubMed

    Mou, Yun; Huang, Po-Ssu; Thomas, Leonard M; Mayo, Stephen L

    2015-08-14

    In standard implementations of computational protein design, a positive-design approach is used to predict sequences that will be stable on a given backbone structure. Possible competing states are typically not considered, primarily because appropriate structural models are not available. One potential competing state, the domain-swapped dimer, is especially compelling because it is often nearly identical with its monomeric counterpart, differing by just a few mutations in a hinge region. Molecular dynamics (MD) simulations provide a computational method to sample different conformational states of a structure. Here, we tested whether MD simulations could be used as a post-design screening tool to identify sequence mutations leading to domain-swapped dimers. We hypothesized that a successful computationally designed sequence would have backbone structure and dynamics characteristics similar to that of the input structure and that, in contrast, domain-swapped dimers would exhibit increased backbone flexibility and/or altered structure in the hinge-loop region to accommodate the large conformational change required for domain swapping. While attempting to engineer a homodimer from a 51-amino-acid fragment of the monomeric protein engrailed homeodomain (ENH), we had instead generated a domain-swapped dimer (ENH_DsD). MD simulations on these proteins showed increased B-factors derived from MD simulation in the hinge loop of the ENH_DsD domain-swapped dimer relative to monomeric ENH. Two point mutants of ENH_DsD designed to recover the monomeric fold were then tested with an MD simulation protocol. The MD simulations suggested that one of these mutants would adopt the target monomeric structure, which was subsequently confirmed by X-ray crystallography.

  20. Specific Protein-Protein Interaction between Basic Helix-Loop-Helix Transcription Factors and Homeoproteins of the Pitx Family

    PubMed Central

    Poulin, Gino; Lebel, Mélanie; Chamberland, Michel; Paradis, Francois W.; Drouin, Jacques

    2000-01-01

    Homeoproteins and basic helix-loop-helix (bHLH) transcription factors are known for their critical role in development and cellular differentiation. The pituitary pro-opiomelanocortin (POMC) gene is a target for factors of both families. Indeed, pituitary-specific transcription of POMC depends on the action of the homeodomain-containing transcription factor Pitx1 and of bHLH heterodimers containing NeuroD1. We now show lineage-restricted expression of NeuroD1 in pituitary corticotroph cells and a direct physical interaction between bHLH heterodimers and Pitx1 that results in transcriptional synergism. The interaction between the bHLH and homeodomains is restricted to ubiquitous (class A) bHLH and to the Pitx subfamily. Since bHLH heterodimers interact with Pitx factors through their ubiquitous moiety, this mechanism may be implicated in other developmental processes involving bHLH factors, such as neurogenesis and myogenesis. PMID:10848608

  1. Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis1[OPEN

    PubMed Central

    Hsiao, An-Shan; Xue, Yan

    2017-01-01

    Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. PMID:28500265

  2. The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity

    PubMed Central

    Teyssier, Catherine; Déméné, Hélène; Carvalho, João E.; Bird, Louise E.; Lebedev, Andrey; Fattori, Juliana; Schubert, Michael; Dumas, Christian; Bourguet, William; le Maire, Albane

    2016-01-01

    Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism. PMID:27812132

  3. Direct regulatory interaction of the eyeless protein with an eye-specific enhancer in the sine oculis gene during eye induction in Drosophila.

    PubMed

    Niimi, T; Seimiya, M; Kloter, U; Flister, S; Gehring, W J

    1999-05-01

    The Pax-6 gene encodes a transcription factor with two DNA-binding domains, a paired and a homeodomain, and is expressed during eye morphogenesis and development of the nervous system. Pax-6 homologs have been isolated from a wide variety of organisms ranging from flatworms to humans. Since loss-of-function mutants in insects and mammals lead to an eyeless phenotype and Pax-6 orthologs from distantly related species are capable of inducing ectopic eyes in Drosophila, we have proposed that Pax-6 is a universal master control gene for eye morphogenesis. To determine the extent of evolutionary conservation of the eye morphogenetic pathway, we have begun to identify subordinate target genes of Pax-6. Previously we have shown that expression of two genes, sine oculis (so) and eyes absent (eya), is induced by eyeless (ey), the Pax-6 homolog of Drosophila. Here we present evidence from ectopic expression studies in transgenic flies, from transcription activation studies in yeast, and from gel shift assays in vitro that the EY protein activates transcription of sine oculis by direct interaction with an eye-specific enhancer in the long intron of the so gene.

  4. Detecting protein-protein interactions using Renilla luciferase fusion proteins.

    PubMed

    Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

    2002-11-01

    We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

  5. The histone demethylase PHF8 is an oncogenic protein in human non-small cell lung cancer

    SciTech Connect

    Shen, Yuzhou; Pan, Xufeng; Zhao, Heng

    2014-08-15

    Highlights: • PHF8 overexpresses in human NSCLC and predicts poor survival. • PHF8 regulates lung cancer cell growth and transformation. • PHF8 regulates apoptosis in human lung cancer cells. • PHF8 promotes miR-21 expression in human lung cancer. • MiR-21 is critically essential for PHF8 function in human lung cancer cells. - Abstract: PHF8 is a JmjC domain-containing protein and erases repressive histone marks including H4K20me1 and H3K9me1/2. It binds to H3K4me3, an active histone mark usually located at transcription start sites (TSSs), through its plant homeo-domain, and is thus recruited and enriched in gene promoters. PHF8 is involved in the development of several types of cancer, including leukemia, prostate cancer, and esophageal squamous cell carcinoma. Herein we report that PHF8 is an oncogenic protein in human non-small cell lung cancer (NSCLC). PHF8 is up-regulated in human NSCLC tissues, and high PHF8 expression predicts poor survival. Our in vitro and in vivo evidence demonstrate that PHF8 regulates lung cancer cell proliferation and cellular transformation. We found that PHF8 knockdown induces DNA damage and apoptosis in lung cancer cells. PHF8 promotes miR-21 expression in human lung cancer, and miR-21 knockdown blocks the effects of PHF8 on proliferation and apoptosis of lung cancer cells. In summary, PHF8 promotes lung cancer cell growth and survival by regulating miR-21.

  6. Regional localization of the CCAAT displacement protein gene (CUTL1) to 7q22 by analysis of somatic cell hybrids

    SciTech Connect

    Scherer, S.W.; Tsui, L.C. The Hospital for Sick Children, Toronto, Ontario ); Neufeld, E.J.; Lievens, P.M.J. Dana Farber Cancer Inst., Cambridge, MA Harvard Medical School, Cambridge, MA ); Orkin, S.H. Dana Farber Cancer Inst., Cambridge, MA Harvard Medical School, Cambridge, MA Howard Hughes Medical Inst., Cambridge, MA ); Kim, J. )

    1993-03-01

    CCAAT displacement protein (CDP) is a putative repressor of tissue-specific gene expression, implicated in regulation of expression of the human myeloid-specific gene, gp91-phox. The cDNA sequence of the human gene predicts a polypeptide of 1506 amino acids, with a homeodomain and three repeated motifs closely homologous to cut, a protein known to specify the cell fate of peripheral neurons and other tissues during embryonic development of Drosophila. Therefore, the human gene locus for CDP has been named CUTL1, for cut-like 1. Preliminary data revealed that CUTL1 mapped to human chromosome 7 (E.J.N., P.M.-J.L., and S.H.O., unpublished results). Since diseases with abnormalities of myeloid differentitation often have aberrations of chromosome 7, especially in patients previously exposed to mutagenic agents or radiation therapy, the cell-type specificity of CDP makes it an attractive candidate for involvement in these conditions. As a first step in addressing this question, we have examined the regional localization of CUTL1 using a panel of rodent-human somatic cell hybrids containing various portions of chromosome 7. The results of our hybridization analysis are summarized. The regional localization of CUTL1 to 7q22 was based primarily on the negative hybridization signal with the human/hamster hybrid 1EF2/3/K017 (7cen-q21) and the positive result with 1CF2/5/K016 (7cen-q22). Since no hybridization signal was detected in GM1059Rag5 (a human-mouse hybrid carrying a single human chromosome 7 with an interstitial deletion 7pter-q22:q32-qter) and 2068Rag22-2 (7qter-q22), the regional assignment of CUTL1 could be narrowed to the distal boundary of 7q22.

  7. The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

    PubMed Central

    Liu, J; Bramblett, D; Zhu, Q; Lozano, M; Kobayashi, R; Ross, S R; Dudley, J P

    1997-01-01

    The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice

  8. Interaction entropy for protein-protein binding

    NASA Astrophysics Data System (ADS)

    Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

    2017-03-01

    Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

  9. DNA-binding proteins and evolution of transcription regulation in the archaea.

    PubMed

    Aravind, L; Koonin, E V

    1999-12-01

    Likely DNA-binding domains in archaeal proteins were analyzed using sequence profile methods and available structural information. It is shown that all archaea encode a large number of proteins containing the helix-turn-helix (HTH) DNA-binding domains whose sequences are much more similar to bacterial HTH domains than to eukaryotic ones, such as the PAIRED, POU and homeodomains. The predominant class of HTH domains in archaea is the winged-HTH domain. The number and diversity of HTH domains in archaea is comparable to that seen in bacteria. The HTH domain in archaea combines with a variety of other domains that include replication system components, such as MCM proteins, translation system components, such as the alpha-subunit of phenyl-alanyl-tRNA synthetase, and several metabolic enzymes. The majority of the archaeal HTH-containing proteins are predicted to be gene/operon-specific transcriptional regulators. This apparent bacterial-type mode of transcription regulation is in sharp contrast to the eukaryote-like layout of the core transcription machinery in the archaea. In addition to the predicted bacterial-type transcriptional regulators, the HTH domain is conserved in archaeal and eukaryotic core transcription factors, such as TFIIB, TFIIE-alpha and MBF1. MBF1 is the only highly conserved, classical HTH domain that is vertically inherited in all archaea and eukaryotes. In contrast, while eukaryotic TFIIB and TFIIE-alpha possess forms of the HTH domain that are divergent in sequence, their archaeal counterparts contain typical HTH domains. It is shown that, besides the HTH domain, archaea encode unexpectedly large numbers of two other predicted DNA-binding domains, namely the Arc/MetJ domain and the Zn-ribbon. The core transcription regulators in archaea and eukaryotes (TFIIB/TFB, TFIIE-alpha and MBF1) and in bacteria (the sigma factors) share no similarity beyond the presence of distinct HTH domains. Thus HTH domains might have been independently recruited for

  10. Learning about Proteins

    MedlinePlus

    ... What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A ... the foods you eat. continue Different Kinds of Protein Protein from animal sources, such as meat and ...

  11. Efficient differentiation of murine embryonic stem cells requires the binding of CXXC finger protein 1 to DNA or methylated histone H3-Lys4.

    PubMed

    Mahadevan, Jyothi; Skalnik, David G

    2016-12-05

    Mammalian CXXC finger protein 1 (Cfp1) is a DNA-binding protein that is a component of the Setd1 histone methyltransferase complexes and is a critical epigenetic regulator of both histone and cytosine methylation. Murine embryonic stem (ES) cells lacking Cfp1 exhibit a loss of histone H3-Lys4 tri-methylation (H3K4me3) at many CpG islands, and a mis-localization of this epigenetic mark to heterochromatic sub-nuclear domains. Furthermore, these cells fail to undergo cellular differentiation in vitro. These defects are rescued upon introduction of a Cfp1-expression vector. Cfp1 contains an N-terminal plant homeodomain (PHD), a motif frequently observed in chromatin associated proteins that functions as a reader module of histone marks. Here, we report that the Cfp1 PHD domain directly and specifically binds to histone H3K4me1/me2/me3 marks. Introduction of individual mutations at key Cfp1 PHD residues (Y28, D44, or W49) ablates this histone interaction both in vitro and in vivo. The W49A point mutation does not affect the ability of Cfp1 to rescue appropriate restriction of histone H3K4me3 to euchromatic sub-nuclear domains or in vitro cellular differentiation in Cfp1-null ES cells. Similarly, a mutated form of Cfp1 that lacks DNA-binding activity (C169A) rescues in vitro cellular differentiation. However, rescue of Cfp1-null ES cells with a double mutant form of Cfp1 (W49A, C169A) results in partially defective in vitro differentiation. These data define the Cfp1 PHD domain as a reader of histone H3K4me marks and provide evidence that this activity is involved in the regulation of lineage commitment in ES cells.

  12. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  13. The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

    PubMed Central

    Cavalieri, Vincenzo; Melfi, Raffaella; Spinelli, Giovanni

    2013-01-01

    Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this

  14. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  15. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  16. PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

    PubMed Central

    Zhang, Tiejun; Park, Kyeong Ah; Li, Yuwen; Byun, Hee Sun; Jeon, Juhee; Lee, Yoonjung; Hong, Jang Hee; Kim, Jin Man; Huang, Song-Mei; Choi, Seung-Won; Kim, Sun-Hwan; Sohn, Kyung-Cheol; Ro, Hyunju; Lee, Ji Hoon; Lu, Tao; Stark, George R.; Shen, Han-Ming; Liu, Zheng-gang; Park, Jongsun; Hur, Gang Min

    2014-01-01

    Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that plant homeodomain finger protein 20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In plant homeodomain finger protein 20-overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between plant homeodomain finger protein 20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that plant homeodomain finger protein 20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies plant homeodomain finger protein 20 as a novel regulator of NF-κB activation and suggests that elevated expression of plant homeodomain finger protein 20 may drive constitutive NF-κB activation in some cancers. PMID:23797602

  17. Direct and Indirect Targets of the E2A-PBX1 Leukemia-Specific Fusion Protein

    PubMed Central

    Diakos, Christofer; Xiao, Yuanyuan; Zheng, Shichun; Kager, Leo; Dworzak, Michael; Wiemels, Joseph L.

    2014-01-01

    E2A-PBX1 is expressed as a result of the t(1;19) chromosomal translocation in nearly 5% of cases of childhood acute lymphoblastic leukemia. The E2A-PBX1 chimeric transcription factor contains the N-terminal transactivation domain of E2A (TCF3) fused to the C-terminal DNA-binding homeodomain of PBX1. While there is no doubt of its oncogenic potential, the mechanisms of E2A-PBX1-mediated pre-B cell transformation and the nature of direct E2A-PBX1 target genes and pathways remain largely unknown. Herein we used chromatin immunoprecipitation assays (ChIP-chip) to identify direct targets of E2A-PBX1, and we used gene expression arrays of siRNA E2A-PBX1-silenced cells to evaluate changes in expression induced by the fusion protein. Combined ChIP-chip and expression data analysis gave rise to direct and functional targets of E2A-PBX1. Further we observe that the set of ChIP-chip identified E2A-PBX1 targets show a collective down-regulation trend in the E2A-PBX1 silenced samples compared to controls suggesting an activating role of this fusion transcription factor. Our data suggest that the expression of the E2A-PBX1 fusion gene interferes with key regulatory pathways and functions of hematopoietic biology. Among these are members of the WNT and apoptosis/cell cycle control pathways, and thus may comprise an essential driving force for the propagation and maintenance of the leukemic phenotype. These findings may also provide evidence of potentially attractive therapeutic targets. PMID:24503810

  18. Germostatin resistance locus 1 encodes a PHD finger protein involved in auxin-mediated seed dormancy and germination.

    PubMed

    Ye, Yajin; Gong, Ziying; Lu, Xiao; Miao, Deyan; Shi, Jianmin; Lu, Juan; Zhao, Yang

    2016-01-01

    Seed dormancy and germination are important physiological processes during the life cycle of a seed plant. Recently, auxin has been characterized as a positive regulator that functions during seed dormancy and as a negative regulator during germination. Through chemical genetic screenings, we have identified a small molecule, germostatin (GS), which effectively inhibits seed germination in Arabidopsis. GSR1 (germostatin resistance locus 1) encodes a tandem plant homeodomain (PHD) finger protein, identified by screening GS-resistant mutants. Certain PHD fingers of GSR1 are capable of binding unmethylated H3K4, which has been reported as an epigenetic mark of gene transcriptional repression. Biochemical studies show that GSR1 physically interacts with the transcriptional repressor ARF16 and attenuates the intensity of interaction of IAA17/ARF16 by directly interacting with IAA17 to release ARF16. Further results show that axr3-1, arf10 arf16 are hyposensitive to GS, and gsr1 not only resists auxin-mediated inhibition of seed germination but also displays decreased dormancy. We therefore propose that GSR1 may form a co-repressor with ARF16 to regulate seed germination. Besides promoting auxin biosynthesis via upregulating expression of YUCCA1, GS also enhances auxin responses by inducing degradation of DΙΙ-VENUS and upregulating expression of DR5-GFP. In summary, we identified GSR1 as a member of the auxin-mediated seed germination genetic network, and GS, a small non-auxin molecule that specifically acts on auxin-mediated seed germination.

  19. Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

    SciTech Connect

    Wang, Shucai; Chang, Ying; Guo, Jianjun; Zeng, Qingning; Ellis, Brian; Chen, Jay

    2011-01-01

    BACKGROUND: The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. CONCLUSIONS/SIGNIFICANCE: Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a

  20. Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

    PubMed Central

    Guo, Jianjun; Zeng, Qingning; Ellis, Brian E.; Chen, Jin-Gui

    2011-01-01

    Background The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. Methodology/Principal Findings We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. Conclusions/Significance Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a

  1. Arabidopsis ovate family proteins, a novel transcriptional repressor family, control multiple aspects of plant growth and development.

    PubMed

    Wang, Shucai; Chang, Ying; Guo, Jianjun; Zeng, Qingning; Ellis, Brian E; Chen, Jin-Gui

    2011-01-01

    The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a previously unknown transcriptional repressor family, and revealed

  2. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  3. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  4. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  5. Protein and Heart Health

    MedlinePlus

    ... It Works Healthy Workplace Food and Beverage Toolkit Protein and Heart Health Updated:May 5,2015 Protein ... said. What’s the harm in getting too much protein? The main problem is that often the extra ...

  6. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  7. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.

  8. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  9. Protein Structure Prediction by Protein Threading

    NASA Astrophysics Data System (ADS)

    Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

    The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

  10. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  11. Whey protein fractionation

    USDA-ARS?s Scientific Manuscript database

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  12. Mirror Image Proteins

    PubMed Central

    Zhao, Le; Lu, Wuyuan

    2017-01-01

    Proteins composed entirely of unnatural D-amino acids and the achiral amino acid glycine are mirror image forms of their native L-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image D-proteins, enabling protein research to be conducted through “the looking glass” and in a way previously unattainable. D-proteins can facilitate structure determination of their native L-forms that are difficult to crystallize (racemic X-ray crystallography); D-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior D-peptide/D-protein therapeutics (mirror image phage display); D-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology. PMID:25282524

  13. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  14. Molecular modelling of protein-protein/protein-solvent interactions

    NASA Astrophysics Data System (ADS)

    Luchko, Tyler

    The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule

  15. Combinatorial protein reagents to manipulate protein function.

    PubMed

    Colas, P

    2000-02-01

    The design and use of combinatorial protein libraries has become a fast moving field in molecular biology. Different experimental systems supporting various selection schemes are now available. The latest breakthroughs include evolutionary experiments to improve existing binding surfaces, selections of homodimerizing peptides, the use of peptide aptamers to disrupt protein interactions inside living cells, and functional selections of aptamers to probe regulatory networks.

  16. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  17. PERSISTENT TAPETAL CELL1 Encodes a PHD-Finger Protein That Is Required for Tapetal Cell Death and Pollen Development in Rice1[C][W][OA

    PubMed Central

    Li, Hui; Yuan, Zheng; Vizcay-Barrena, Gema; Yang, Caiyun; Liang, Wanqi; Zong, Jie; Wilson, Zoe A.; Zhang, Dabing

    2011-01-01

    In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development. PMID:21515697

  18. NBS-LRR Protein Pik-H4 Interacts with OsBIHD1 to Balance Rice Blast Resistance and Growth by Coordinating Ethylene-Brassinosteroid Pathway

    PubMed Central

    Liu, Hao; Dong, Shuangyu; Gu, Fengwei; Liu, Wei; Yang, Guili; Huang, Ming; Xiao, Wuming; Liu, Yongzhu; Guo, Tao; Wang, Hui; Chen, Zhiqiang; Wang, Jiafeng

    2017-01-01

    The regulation of innate immunity and plant growth, along with the trade-off between them, affects the defense and recovery mechanisms of the plant after it is attacked by pathogens. Although it is known that hormonal crosstalk plays a major role in regulating interaction of plant growth and PAMP-triggered immunity, the relationship between plant growth and effector-triggered immunity (ETI) remains unclear. In a large-scale yeast two-hybrid screening for Pik-H4-interacting proteins, a homeodomain transcription factor OsBIHD1 was identified, which is previously known to function in biotic and abiotic stress responses. The knockout of OsBIHD1 in rice lines carrying Pik-H4 largely compromised the resistance of the rice lines to Magnaporthe oryzae, the fungus that causes rice blast. While overexpression of OsBIHD1 resulted in enhanced expression of the pathogenesis-related (PR) and ethylene (ET) synthesis genes. Moreover, OsBIHD1 was also found to directly bind to the promoter region of ethylene-synthesis enzyme OsACO3. In addition, OsBIHD1 overexpression or deficiency provoked dwarfism and reduced brassinosteroid (BR) insensitivity through repressing the expression of several critical genes involved in BR biosynthesis and BR signaling. During M. oryzae infection, transcript levels of the crucial BR catabolic genes (CYP734A2, CYP734A4, and CYP734A6) were significantly up-regulated in OsBIHD1-OX plants. Furthermore, OsBIHD1 was found to be capable of binding to the sequence-specific cis-elements on the promoters of CYP734A2 to suppress the plant growth under fungal invasion. Our results collectively suggest a model that OsBIHD1 is required for Pik-H4-mediated blast resistance through modulating the trade-off between resistance and growth by coordinating brassinosteroid-ethylene pathway. PMID:28220140

  19. 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.

  20. PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice.

    PubMed

    Li, Hui; Yuan, Zheng; Vizcay-Barrena, Gema; Yang, Caiyun; Liang, Wanqi; Zong, Jie; Wilson, Zoe A; Zhang, Dabing

    2011-06-01

    In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.

  1. NBS-LRR Protein Pik-H4 Interacts with OsBIHD1 to Balance Rice Blast Resistance and Growth by Coordinating Ethylene-Brassinosteroid Pathway.

    PubMed

    Liu, Hao; Dong, Shuangyu; Gu, Fengwei; Liu, Wei; Yang, Guili; Huang, Ming; Xiao, Wuming; Liu, Yongzhu; Guo, Tao; Wang, Hui; Chen, Zhiqiang; Wang, Jiafeng

    2017-01-01

    The regulation of innate immunity and plant growth, along with the trade-off between them, affects the defense and recovery mechanisms of the plant after it is attacked by pathogens. Although it is known that hormonal crosstalk plays a major role in regulating interaction of plant growth and PAMP-triggered immunity, the relationship between plant growth and effector-triggered immunity (ETI) remains unclear. In a large-scale yeast two-hybrid screening for Pik-H4-interacting proteins, a homeodomain transcription factor OsBIHD1 was identified, which is previously known to function in biotic and abiotic stress responses. The knockout of OsBIHD1 in rice lines carrying Pik-H4 largely compromised the resistance of the rice lines to Magnaporthe oryzae, the fungus that causes rice blast. While overexpression of OsBIHD1 resulted in enhanced expression of the pathogenesis-related (PR) and ethylene (ET) synthesis genes. Moreover, OsBIHD1 was also found to directly bind to the promoter region of ethylene-synthesis enzyme OsACO3. In addition, OsBIHD1 overexpression or deficiency provoked dwarfism and reduced brassinosteroid (BR) insensitivity through repressing the expression of several critical genes involved in BR biosynthesis and BR signaling. During M. oryzae infection, transcript levels of the crucial BR catabolic genes (CYP734A2, CYP734A4, and CYP734A6) were significantly up-regulated in OsBIHD1-OX plants. Furthermore, OsBIHD1 was found to be capable of binding to the sequence-specific cis-elements on the promoters of CYP734A2 to suppress the plant growth under fungal invasion. Our results collectively suggest a model that OsBIHD1 is required for Pik-H4-mediated blast resistance through modulating the trade-off between resistance and growth by coordinating brassinosteroid-ethylene pathway.

  2. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  3. Conformational changes induced in Hoxb-8/Pbx-1 heterodimers in solution and upon interaction with specific DNA.

    PubMed Central

    Sánchez, M; Jennings, P A; Murre, C

    1997-01-01

    Two classes of homeodomain proteins, Hox and Pbx gene products, have the ability to bind cooperatively to DNA. In Hox proteins, the homeodomain and a highly conserved hexapeptide are required for cooperative DNA binding. In Pbx, the homeodomain and a region immediately C terminal of the homeodomain are essential for cooperativity. Using fluorescence and circular dichroism spectroscopy, we demonstrated that Hox and Pbx proteins interact in the absence of DNA. The interaction in solution is accompanied by conformational changes. Furthermore, upon interaction with specific DNA, additional conformational changes are induced in the Pbx-1/Hoxb-8 heterodimer. These data indicate that prior to DNA binding, Hox-Pbx interaction in solution is accompanied by structural alterations. We propose that these conformational changes modulate the DNA binding properties of these proteins, ultimately resulting in cooperative DNA binding. PMID:9271414

  4. Hydrodynamic effects in proteins.

    PubMed

    Szymczak, Piotr; Cieplak, Marek

    2011-01-26

    Experimental and numerical results pertaining to flow-induced effects in proteins are reviewed. Special emphasis is placed on shear-induced unfolding and on the role of solvent mediated hydrodynamic interactions in the conformational transitions in proteins.

  5. Learning about Proteins

    MedlinePlus

    ... body, and protecting you from disease. All About Amino Acids When you eat foods that contain protein, the ... called amino (say: uh-MEE-no) acids. The amino acids then can be reused to make the proteins ...

  6. Protein C blood test

    MedlinePlus

    ... a normal substance in the body that prevents blood clotting. A blood test can be done to see ... history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with ...

  7. Protein S blood test

    MedlinePlus

    ... a normal substance in your body that prevents blood clotting. A blood test can be done to see ... family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with ...

  8. Protein and older adults.

    PubMed

    Chernoff, Ronni

    2004-12-01

    Body composition changes as people get older. One of the noteworthy alterations is the reduction in total body protein. A decrease in skeletal muscle is the most noticeable manifestation of this change but there is also a reduction in other physiologic proteins such as organ tissue, blood components, and immune bodies as well as declines in total body potassium and water. This contributes to impaired wound healing, loss of skin elasticity, and an inability to fight infection. The recommended dietary allowance (RDA) for adults for protein is 0.8 grams of protein per kilogram of body weight. Protein tissue accounts for 30% of whole-body protein turnover but that rate declines to 20% or less by age 70. The result of this phenomenon is that older adults require more protein/kilogram body weight than do younger adults. Recently, it has become clear that the requirement for exogenous protein is at least 1.0 gram/kilogram body weight. Adequate dietary intake of protein may be more difficult for older adults to obtain. Dietary animal protein is the primary source of high biological value protein, iron, vitamin B(12), folic acid, biotin and other essential nutrients. In fact, egg protein is the standard against which all other proteins are compared. Compared to other high-quality protein sources like meat, poultry and seafood, eggs are the least expensive. The importance of dietary protein cannot be underestimated in the diets of older adults; inadequate protein intake contributes to a decrease in reserve capacity, increased skin fragility, decreased immune function, poorer healing, and longer recuperation from illness.

  9. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth

    2004-01-01

    Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

  10. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  11. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  12. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  13. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  14. Texturized dairy proteins

    USDA-ARS?s Scientific Manuscript database

    Dairy proteins are amenable to structural modifications induced by high temperature, shear and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey prote...

  15. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  16. SAMPyling Proteins in Archaea

    PubMed Central

    Darwin, K. Heran; Hofmann, Kay

    2010-01-01

    For some time post-translational small protein modifications were found only in eukaryotes; much later, such modifications were identified in some species of bacteria. The recent discovery of ubiquitin-like proteins that form polymeric chains and covalently modify proteins in archaea finally closes the evolutionary gap among the domains of life. PMID:20547064

  17. The E5 Proteins

    PubMed Central

    DiMaio, Daniel; Petti, Lisa

    2013-01-01

    The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 is the PDGF β receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis. PMID:23731971

  18. Protein-protein interactions in multienzyme megasynthetases.

    PubMed

    Weissman, Kira J; Müller, Rolf

    2008-04-14

    The multienzyme polyketide synthases (PKSs), nonribosomal polypeptide synthetases (NRPSs), and their hybrids are responsible for the construction in bacteria of numerous natural products of clinical value. These systems generate high structural complexity by using a simple biosynthetic logic--that of the assembly line. Each of the individual steps in building the metabolites is designated to an independently folded domain within gigantic polypeptides. The domains are clustered into functional modules, and the modules are strung out along the proteins in the order in which they act. Every metabolite results, therefore, from the successive action of up to 100 individual catalysts. Despite the conceptual simplicity of this division-of-labor organization, we are only beginning to decipher the molecular details of the numerous protein-protein interactions that support assembly-line biosynthesis, and which are critical to attempts to re-engineer these systems as a tool in drug discovery. This review aims to summarize the state of knowledge about several aspects of protein-protein interactions, including current architectural models for PKS and NRPS systems, the central role of carrier proteins, and the structural basis for intersubunit recognition.

  19. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  20. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  1. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  2. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  3. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  4. Forces Stabilizing Proteins

    PubMed Central

    Pace, C. Nick; Scholtz, J. Martin; Grimsley, Gerald R.

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  5. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  6. Protein and vegetarian diets.

    PubMed

    Marsh, Kate A; Munn, Elizabeth A; Baines, Surinder K

    2013-08-19

    A vegetarian diet can easily meet human dietary protein requirements as long as energy needs are met and a variety of foods are eaten. Vegetarians should obtain protein from a variety of plant sources, including legumes, soy products, grains, nuts and seeds. Eggs and dairy products also provide protein for those following a lacto-ovo-vegetarian diet. There is no need to consciously combine different plant proteins at each meal as long as a variety of foods are eaten from day to day, because the human body maintains a pool of amino acids which can be used to complement dietary protein. The consumption of plant proteins rather than animal proteins by vegetarians may contribute to their reduced risk of chronic diseases such as diabetes and heart disease.

  7. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  8. Protein Folding: Detailed Models

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    Proteins play a fundamental role in biology. With their ability to perform numerous biological roles, including acting as catalysts, antibodies, and molecular signals, proteins today realize many of the goals that modern nanotechnology aspires to. However, before proteins can carry out these remarkable molecular functions, they must perform another amazing feat — they must assemble themselves. This process of protein self-assembly into a particular shape, or "fold" is called protein folding. Due to the importance of the folded state in the biological activity of proteins, recent interest from misfolding related diseases [1], as well as a fascination of just how this process occurs [2-4], there has been much work performed in order to unravel the mechanism of protein folding [5].

  9. [Atypical ubiquitination of proteins].

    PubMed

    Buneeva, O A; Medvedev, A E

    2016-07-01

    Ubiquitination is a type of posttranslational modification of intracellular proteins characterized by covalent attachment of one (monoubiquitination) or several (polyubiquitination) of ubiquitin molecules to target proteins. In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins not necessarily targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes and altered polyubiquitination of certain proteins is crucial for development of serious diseases.

  10. The Protein Folding Problem

    PubMed Central

    Dill, Ken A.; Ozkan, S. Banu; Shell, M. Scott; Weikl, Thomas R.

    2008-01-01

    The “protein folding problem” consists of three closely related puzzles: (a) What is the folding code? (b) What is the folding mechanism? (c) Can we predict the native structure of a protein from its amino acid sequence? Once regarded as a grand challenge, protein folding has seen great progress in recent years. Now, foldable proteins and nonbiological polymers are being designed routinely and moving toward successful applications. The structures of small proteins are now often well predicted by computer methods. And, there is now a testable explanation for how a protein can fold so quickly: A protein solves its large global optimization problem as a series of smaller local optimization problems, growing and assembling the native structure from peptide fragments, local structures first. PMID:18573083

  11. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  12. Packing in protein cores

    NASA Astrophysics Data System (ADS)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  13. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  14. A rapidly evolving homeobox at the site of a hybrid sterility gene.

    PubMed

    Ting, C T; Tsaur, S C; Wu, M L; Wu, C I

    1998-11-20

    The homeodomain is a DNA binding motif that is usually conserved among diverse taxa. Rapidly evolving homeodomains are thus of interest because their divergence may be associated with speciation. The exact site of the Odysseus (Ods) locus of hybrid male sterility in Drosophila contains such a homeobox gene. In the past half million years, this homeodomain has experienced more amino acid substitutions than it did in the preceding 700 million years; during this period, it has also evolved faster than other parts of the protein or even the introns. Such rapid sequence divergence is driven by positive selection and may contribute to reproductive isolation.

  15. Toxic proteins in plants.

    PubMed

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Reverse Phase Protein Microarrays.

    PubMed

    Baldelli, Elisa; Calvert, Valerie; Hodge, Alex; VanMeter, Amy; Petricoin, Emanuel F; Pierobon, Mariaelena

    2017-01-01

    While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive all cellular functions including proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool for understanding cellular functions.Alteration of the cellular homeostasis driven by elaborate intra- and extracellular interactions has become one of the most studied fields in the era of personalized medicine and targeted therapy. Increasing interest has been focused on developing and improving proteomic technologies that are suitable for analysis of clinical samples. In this context, reverse-phase protein microarrays (RPPA) is a sensitive, quantitative, high-throughput immunoassay for protein analyses of tissue samples, cells, and body fluids.RPPA is well suited for broad proteomic profiling and is capable of capturing protein activation as well as biochemical reactions such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations across hundreds of samples using a limited amount of biological material. For these reasons, RPPA represents a valid tool for protein analyses and generates data that help elucidate the functional signaling architecture through protein-protein interaction and protein activation mapping for the identification of critical nodes for individualized or combinatorial targeted therapy.

  17. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  18. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  19. A modified Lowry protein test for dilute protein solutions

    Treesearch

    Garold F. Gregory; Keith F. Jensen

    1971-01-01

    A modified Lowry protein test for dilute protein solutions modified Lowry protein test was compared with the standard Lowry protein test. The modified test was found to give estimates of protein concentration that were as good as the standard test and has the advange that proteins can be measured in very dilute solutions.

  20. Protein flexibility as a biosignal.

    PubMed

    Zhao, Qinyi

    2010-01-01

    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.