Becker, Peter B.; Workman, Jerry L.
Eukaryotic chromatin is kept flexible and dynamic to respond to environmental, metabolic, and developmental cues through the action of a family of so-called “nucleosome remodeling” ATPases. Consistent with their helicase ancestry, these enzymes experience conformation changes as they bind and hydrolyze ATP. At the same time they interact with DNA and histones, which alters histone–DNA interactions in target nucleosomes. Their action may lead to complete or partial disassembly of nucleosomes, the exchange of histones for variants, the assembly of nucleosomes, or the movement of histone octamers on DNA. “Remodeling” may render DNA sequences accessible to interacting proteins or, conversely, promote packing into tightly folded structures. Remodeling processes participate in every aspect of genome function. Remodeling activities are commonly integrated with other mechanisms such as histone modifications or RNA metabolism to assemble stable, epigenetic states. PMID:24003213
Al-Ani, Gada; Malik, Shuja Shafi; Eastlund, Allen; Briggs, Koan; Fischer, Christopher J
The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction.
The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction. PMID:24898619
Ramachandran, Srinivas; Henikoff, Steven
Precise positioning of nucleosomes around regulatory sites is achieved by the action of chromatin remodelers, which use the energy of ATP to slide, evict or change the composition of nucleosomes. Chromatin remodelers act to bind nucleosomes, disrupt histone-DNA interactions and translocate the DNA around the histone core to reposition nucleosomes. Hence, remodeling is expected to involve nucleosomal intermediates with a structural organization that is distinct from intact nucleosomes. We describe the identification of a partially unwrapped nucleosome structure using methods that map histone-DNA contacts genome-wide. This alternative nucleosome structure is likely formed as an intermediate or by-product during nucleosome remodeling by the RSC complex. Identification of the loss of histone-DNA contacts during chromatin remodeling by RSC in vivo has implications for the regulation of transcriptional initiation.
Yen, Kuangyu; Vinayachandran, Vinesh; Batta, Kiran; Koerber, R. Thomas; Pugh, B. Franklin
How chromatin remodelers cooperate to organize nucleosomes around the start and end of genes is not known. We determined the genome-wide binding of remodeler complexes SWI/SNF, RSC, ISW1a, ISW1b, ISW2, and INO80 to individual nucleosomes in Saccharomyces, and determined their functional contributions to nucleosome positioning through deletion analysis. We applied ultra-high resolution ChIP-exo mapping to Isw2 to determine its sub-nucleosomal orientation and organization on a genomic scale. Remodelers interacted with selected nucleosome positions relative to the start and end of genes, and produced net directionality in moving nucleosomes either away or towards nucleosome-free regions at the 5′ and 3′ ends of genes. Isw2 possessed a sub-nucleosomal organization in accord with biochemical and crystallographic-based models that place its linker binding region within promoters and abutted against Reb1-bound locations. Together these findings reveal a coordinated position-specific approach taken by remodelers to organize genic nucleosomes into arrays. PMID:22726434
Background Eukaryotic chromatin architecture is affected by intrinsic histone-DNA sequence preferences, steric exclusion between nucleosome particles, formation of higher-order structures, and in vivo activity of chromatin remodeling enzymes. Results To disentangle sequence-dependent nucleosome positioning from the other factors, we have created two high-throughput maps of nucleosomes assembled in vitro on genomic DNA from the nematode worm Caenorhabditis elegans. A comparison of in vitro nucleosome positions with those observed in a mixed-stage, mixed-tissue population of C. elegans cells reveals that in vivo sequence preferences are modified on the genomic scale. Indeed, G/C dinucleotides are predicted to be most favorable for nucleosome formation in vitro but not in vivo. Nucleosome sequence read coverage in vivo is distinctly lower in chromosome arms than in central regions; the observed changes in apparent nucleosome sequence specificity, likely due to genome-wide chromatin remodeler activity, contribute to the formation of these megabase-scale chromatin domains. We also observe that the majority of well-positioned in vivo nucleosomes do not occupy thermodynamically favorable sequences observed in vitro. Finally, we find that exons are intrinsically more amenable to nucleosome formation compared to introns. Nucleosome occupancy of introns and exons consistently increases with G/C content in vitro but not in vivo, in agreement with our observation that G/C dinucleotide enrichment does not strongly promote in vivo nucleosome formation. Conclusions Our findings highlight the importance of both sequence specificity and active nucleosome repositioning in creating large-scale chromatin domains, and the antagonistic roles of intrinsic sequence preferences and chromatin remodelers in C. elegans. Sequence read data has been deposited into Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra; accession number SRA050182). Additional data, software and computational
Shi, Jiejun; Zheng, Meizhu; Ye, Youqiong; Li, Min; Chen, Xiaolong; Hu, Xinjie; Sun, Jin; Zhang, Xiaobai; Jiang, Cizhong
ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.
At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome.
Kwon, So Yeon; Grisan, Valentina; Jang, Boyun; Herbert, John; Badenhorst, Paul
NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions. PMID:27046080
Peterson, Craig L
ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states.
Harada, Bryan T; Hwang, William L; Deindl, Sebastian; Chatterjee, Nilanjana; Bartholomew, Blaine; Zhuang, Xiaowei
The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.
Patrick, Kristin L.; Ryan, Colm J.; Xu, Jiewei; Lipp, Jesse J.; Nissen, Kelly E.; Roguev, Assen; Shales, Michael; Krogan, Nevan J.; Guthrie, Christine
Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects—and is affected by—co-transcriptional splicing. PMID:25825871
Patrick, Kristin L; Ryan, Colm J; Xu, Jiewei; Lipp, Jesse J; Nissen, Kelly E; Roguev, Assen; Shales, Michael; Krogan, Nevan J; Guthrie, Christine
Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.
de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Bou Dargham, Daria; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B Franklin; Gérard, Matthieu
ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.
de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Dargham, Daria Bou; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P.; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B. Franklin; Gérard, Matthieu
Summary ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers1–3 target specific nucleosomes to regulate transcription is unclear. Here, we present genome-wide remodeller-nucleosome interaction profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank MNase-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites (TSSs) are nevertheless chromatinized with non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and modifications (H3K4me3 and H3K27ac). RNA polymerase (pol) II therefore navigates hundreds of bp of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3′ end of the NFR. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers play either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs. PMID:26814966
Li, Ming; Hada, Arjan; Sen, Payel; Olufemi, Lola; Hall, Michael A; Smith, Benjamin Y; Forth, Scott; McKnight, Jeffrey N; Patel, Ashok; Bowman, Gregory D; Bartholomew, Blaine; Wang, Michelle D
The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.
Lieleg, Corinna; Ketterer, Philip; Nuebler, Johannes; Ludwigsen, Johanna; Gerland, Ulrich; Dietz, Hendrik
Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms. PMID:25733687
Lieleg, Corinna; Ketterer, Philip; Nuebler, Johannes; Ludwigsen, Johanna; Gerland, Ulrich; Dietz, Hendrik; Mueller-Planitz, Felix; Korber, Philipp
Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the Hand-Sant-Slide (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.
Tolkunov, Denis; Zawadzki, Karl A; Singer, Cara; Elfving, Nils; Morozov, Alexandre V; Broach, James R
Most promoters in yeast contain a nucleosome-depleted region (NDR), but the mechanisms by which NDRs are established and maintained in vivo are currently unclear. We have examined how genome-wide nucleosome placement is altered in the absence of two distinct types of nucleosome remodeling activity. In mutants of both SNF2, which encodes the ATPase component of the Swi/Snf remodeling complex, and ASF1, which encodes a histone chaperone, distinct sets of gene promoters carry excess nucleosomes in their NDRs relative to wild-type. In snf2 mutants, excess promoter nucleosomes correlate with reduced gene expression. In both mutants, the excess nucleosomes occupy DNA sequences that are energetically less favorable for nucleosome formation, indicating that intrinsic histone-DNA interactions are not sufficient for nucleosome positioning in vivo, and that Snf2 and Asf1 promote thermodynamic equilibration of nucleosomal arrays. Cells lacking SNF2 or ASF1 still accomplish the changes in promoter nucleosome structure associated with large-scale transcriptional reprogramming. However, chromatin reorganization in the mutants is reduced in extent compared to wild-type cells, even though transcriptional changes proceed normally. In summary, active remodeling is required for distributing nucleosomes to energetically favorable positions in vivo and for reorganizing chromatin in response to changes in transcriptional activity.
Sala, Anna; Toto, Maria; Pinello, Luca; Gabriele, Alessandra; Di Benedetto, Valeria; Ingrassia, Antonia M R; Lo Bosco, Giosuè; Di Gesù, Vito; Giancarlo, Raffaele; Corona, Davide F V
The evolutionarily conserved ATP-dependent nucleosome remodelling factor ISWI can space nucleosomes affecting a variety of nuclear processes. In Drosophila, loss of ISWI leads to global transcriptional defects and to dramatic alterations in higher-order chromatin structure, especially on the male X chromosome. In order to understand if chromatin condensation and gene expression defects, observed in ISWI mutants, are directly correlated with ISWI nucleosome spacing activity, we conducted a genome-wide survey of ISWI binding and nucleosome positioning in wild-type and ISWI mutant chromatin. Our analysis revealed that ISWI binds both genic and intergenic regions. Remarkably, we found that ISWI binds genes near their promoters causing specific alterations in nucleosome positioning at the level of the Transcription Start Site, providing an important insights in understanding ISWI role in higher eukaryote transcriptional regulation. Interestingly, differences in nucleosome spacing, between wild-type and ISWI mutant chromatin, tend to accumulate on the X chromosome for all ISWI-bound genes analysed. Our study shows how in higher eukaryotes the activity of the evolutionarily conserved nucleosome remodelling factor ISWI regulates gene expression and chromosome organization genome-wide. PMID:21448136
Parmar, Jyotsana J; Marko, John F; Padinhateeri, Ranjith
We investigate how DNA sequence, ATP-dependent chromatin remodeling and nucleosome-depleted 'barriers' co-operate to determine the kinetics of nucleosome organization, in a stochastic model of nucleosome positioning and dynamics. We find that 'statistical' positioning of nucleosomes against 'barriers', hypothesized to control chromatin structure near transcription start sites, requires active remodeling and therefore cannot be described using equilibrium statistical mechanics. We show that, unlike steady-state occupancy, DNA site exposure kinetics near a barrier is dominated by DNA sequence rather than by proximity to the barrier itself. The timescale for formation of positioning patterns near barriers is proportional to the timescale for active nucleosome eviction. We also show that there are strong gene-to-gene variations in nucleosome positioning near barriers, which are eliminated by averaging over many genes. Our results suggest that measurement of nucleosome kinetics can reveal information about sequence-dependent regulation that is not apparent in steady-state nucleosome occupancy.
Seeber, Andrew; Hauer, Michael; Gasser, Susan M
ATP-dependent nucleosome remodelers use ATP hydrolysis to shift, evict and exchange histone dimers or octamers and have well-established roles in transcription. Earlier work has suggested a role for nucleosome remodelers such as INO80 in double-strand break (DSB) repair. This review will begin with an update on recent studies that explore how remodelers are recruited to DSBs. We then examine their impact on various steps of repair, focusing on resection and the formation of the Rad51-ssDNA nucleofilament. Finally, we will explore new studies that implicate remodelers in the physical movement of chromatin in response to damage.
Lee, Junwoo; Shik Choi, Eun; David Seo, Hogyu; Kang, Keunsoo; Gilmore, Joshua M.; Florens, Laurence; Washburn, Michael P.; Choe, Joonho; Workman, Jerry L.; Lee, Daeyoup
Previous studies have revealed that nucleosomes impede elongation of RNA polymerase II (RNAPII). Recent observations suggest a role for ATP-dependent chromatin remodellers in modulating this process, but direct in vivo evidence for this is unknown. Here using fission yeast, we identify Fun30Fft3 as a chromatin remodeller, which localizes at transcribing regions to promote RNAPII transcription. Fun30Fft3 associates with RNAPII and collaborates with the histone chaperone, FACT, which facilitates RNAPII elongation through chromatin, to induce nucleosome disassembly at transcribing regions during RNAPII transcription. Mutants, resulting in reduced nucleosome-barrier, such as deletion mutants of histones H3/H4 themselves and the genes encoding components of histone deacetylase Clr6 complex II suppress the defects in growth and RNAPII occupancy of cells lacking Fun30Fft3. These data suggest that RNAPII utilizes the chromatin remodeller, Fun30Fft3, to overcome the nucleosome barrier to transcription elongation. PMID:28218250
Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo
The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520
Chaban, Yuriy; Ezeokonkwo, Chukwudi; Chung, Wen-Hsiang; Zhang, Fan; Kornberg, Roger D; Maier-Davis, Barbara; Lorch, Yahli; Asturias, Francisco J
ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict or restructure nucleosomes. A structure of a RSC-nucleosome complex with a nucleosome determined by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into solution as required for remodeling, but the structure of the RSC-nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC-nucleosome structure and published biochemical information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.
Byeon, Boseon; Wang, Wei; Barski, Artem; Ranallo, Ryan T; Bao, Kan; Schones, Dustin E; Zhao, Keji; Wu, Carl; Wu, Wei-Hua
The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.
Yen, Kuangyu; Vinayachandran, Vinesh; Pugh, B Franklin
SWR-C/SWR1 and INO80 are multisubunit complexes that catalyze the deposition and removal, respectively, of histone variant H2A.Z from the first nucleosome at the start of genes. How they target and engage these +1 nucleosomes is unclear. Using ChIP-exo, we identified the subnucleosomal placement of 20 of their subunits across the yeast genome. The Swc2 subunit of SWR-C bound a narrowly defined region in the adjacent nucleosome-free region (NFR), where it positioned the Swr1 subunit over one of two sites of H2A.Z deposition at +1. The genomic binding maps suggest that many subunits have a rather plastic organization that allows subunits to exchange between the two complexes. One outcome of promoting H2A/H2A.Z exchange was an enhanced turnover of entire nucleosomes, thereby creating dynamic chromatin at the start of genes. Our findings provide unifying concepts on how these two opposing chromatin remodeling complexes function selectively at the +1 nucleosome of nearly all genes.
Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions ('genomic code for nucleosome positioning'). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called 'statistical positioning' may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5' ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.
Westenberger, Scott J; Cui, Long; Dharia, Neekesh; Winzeler, Elizabeth; Cui, Liwang
Epigenetic modifications of histones and regulation of chromatin structure have been implicated in regulation of virulence gene families in P. falciparum. To better understand chromatin-mediated gene regulation, we used a high-density oligonucleotide microarray to map the position and enrichment of nucleosomes across the entire genome of P. falciparum at three time points of the intra-erythrocytic developmental cycle (IDC) in vitro. We used an unmodified histone H4 antibody for chromatin immunoprecipitation of nucleosome-bound DNA. We observed generally low nucleosomal occupancy of intergenic regions and higher occupancy of protein coding regions. In contract to the overall small fluctuation of nucleosomal occupancy in most coding regions throughout the IDC, subtelomeric genes encoding surface proteins such as var and rif, as well as some core chromosomal genes such as transcription factors, showed large changes in chromatin structure. Telomeres harbored a region with the highest nucleosomal occupancy of the genome and also exhibited large changes with higher nucleosomal occupancy at schizont stages. While many of these subtelomeric genes were previously shown to be modified by H3K9 trimethylation, we also identified some housekeeping genes in core chromosome regions that showed extensive changes in chromatin structure but do not contain this modification. tRNA and basal transcription factor genes showed low nucleosomal occupancy at all times, suggesting of an open chromatin structure that might be permissive for constitutively high levels of expression. Generally, nucleosomal occupancy was not correlated with the steady-state mRNA levels. Several var genes were exceptions: the var gene with the highest expression level showed the lowest nucleosomal occupancy, and selection of parasites for var2CSA expression resulted in lower nucleosomal occupancy at the var2CSA locus. We identified nucleosome-free regions in intergenic regions that may serve as transcription
Florescu, Ana-Maria; Schiessel, Helmut; Blossey, Ralf
Chromatin structure is dynamically organized by chromatin remodelers, motor protein complexes which move and remove nucleosomes. The regulation of remodeler action has recently been proposed to underlie a kinetic proofreading scheme which combines the recognition of histone-tail states and the ATP-dependent loosening of DNA around nucleosomes. Members of the ISWI-family of remodelers additionally recognize linker length between nucleosomes. Here, we show that the additional proofreading step involving linker length alone is sufficient to promote the formation of regular arrays of nucleosomes. ATP-dependent remodeling by bidirectional motors is shown to reinforce positioning as compared to statistical positioning.
Buning, Ruth; Kropff, Wietske; Martens, Kirsten; van Noort, John
Chromatin, the structure in which DNA is compacted in eukaryotic cells, plays a key role in regulating DNA accessibility. FRET experiments on single nucleosomes, the basic units in chromatin, have revealed a dynamic nucleosome where spontaneous DNA unwrapping from the ends provides access to the nucleosomal DNA. Here we investigated how this DNA breathing is affected by extension of the linker DNA and by the presence of a neighboring nucleosome. We found that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping. Interactions between neighboring nucleosomes are more likely in dinucleosomes spaced by 55 bp of linker DNA than in dinucleosomes spaced by 50 bp of linker DNA. Such increased unwrapping may not only increase the accessibility of nucleosomal DNA in chromatin fibers, it may also be key to folding of nucleosomes into higher order structures.
Buning, Ruth; Kropff, Wietske; Martens, Kirsten; van Noort, John
Chromatin, the structure in which DNA is compacted in eukaryotic cells, plays a key role in regulating DNA accessibility. FRET experiments on single nucleosomes, the basic units in chromatin, have revealed a dynamic nucleosome where spontaneous DNA unwrapping from the ends provides access to the nucleosomal DNA. Here we investigated how this DNA breathing is affected by extension of the linker DNA and by the presence of a neighboring nucleosome. We found that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping. Interactions between neighboring nucleosomes are more likely in dinucleosomes spaced by 55 bp of linker DNA than in dinucleosomes spaced by 50 bp of linker DNA. Such increased unwrapping may not only increase the accessibility of nucleosomal DNA in chromatin fibers, it may also be key to folding of nucleosomes into higher order structures.
Verdaasdonk, Jolien S; Gardner, Ryan; Stephens, Andrew D; Yeh, Elaine; Bloom, Kerry
Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis.
Platt, James L.; Kent, Nicholas A.; Kimmel, Alan R.
Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states. PMID:28330902
The packaging of DNA into nucleosomes imposes obstacles on gene transcription, and histone-modifying and nucleosome-remodeling complexes work in concert to alleviate these obstacles so as to facilitate transcription. Emerging evidence shows that chromatin-associated poly(ADP-ribose) polymerase 1 (PARP-1) and its enzymatic activity facilitate inflammatory gene transcription and modulate the inflammatory response in animal models. However, the molecular mechanisms by which PARP-1 enzymatic activity facilitates transcription are not well understood. Here we show that through an intracellular signaling pathway, lipopolysaccharide (LPS) stimulation induces PARP-1 enzymatic activity and the ADP-ribosylation of histones at transcriptionally active and accessible chromatin regions in macrophages. In vitro DNase I footprinting and restriction endonuclease accessibility assays reveal that histone ADP-ribosylation directly destabilizes histone-DNA interactions in the nucleosome and increases the site accessibility of the nucleosomal DNA to nucleases. Consistent with this, LPS stimulation-induced ADP-ribosylation at the nucleosome-occupied promoters of il-1β, mip-2, and csf2 facilitates NF-κB recruitment and the transcription of these genes in macrophages. Therefore, our data suggest that PARP-1 enzymatic activity facilitates gene transcription through increasing promoter accessibility by histone ADP-ribosylation. PMID:22547677
Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D
AT-rich DNA is concentrated in the nucleosome-free regions (NFRs) associated with transcription start sites of most genes. We tested the hypothesis that AT-rich DNA engenders NFR formation by virtue of its rigidity and consequent exclusion of nucleosomes. We found that the AT-rich sequences present in many NFRs have little effect on the stability of nucleosomes. Rather, these sequences facilitate the removal of nucleosomes by the RSC chromatin remodeling complex. RSC activity is stimulated by AT-rich sequences in nucleosomes and inhibited by competition with AT-rich DNA. RSC may remove NFR nucleosomes without effect on adjacent ORF nucleosomes. Our findings suggest that many NFRs are formed and maintained by an active mechanism involving the ATP-dependent removal of nucleosomes rather than a passive mechanism due to the intrinsic instability of nucleosomes on AT-rich DNA sequences. © 2014 Lorch et al.; Published by Cold Spring Harbor Laboratory Press.
Bowman, Gregory D.
ATP-dependent chromatin remodelers regulate chromatin dynamics by modifying nucleosome positions and occupancy. DNA-dependent processes such as replication and transcription rely on chromatin to faithfully regulate DNA accessibility, yet how chromatin remodelers achieve well-defined nucleosome positioning in vivo is poorly understood. Here, we report a simple method for site-specifically altering nucleosome positions in live cells. By fusing the Chd1 remodeler to the DNA binding domain of the Saccharomyces cerevisiae Ume6 repressor, we have engineered a fusion remodeler that selectively positions nucleosomes on top of adjacent Ume6 binding motifs in a highly predictable and reproducible manner. Positioning of nucleosomes by the fusion remodeler recapitulates closed chromatin structure at Ume6-sensitive genes analogous to the endogenous Isw2 remodeler. Strikingly, highly precise positioning of single founder nucleosomes by either chimeric Chd1-Ume6 or endogenous Isw2 shifts phased chromatin arrays in cooperation with endogenous chromatin remodelers. Our results demonstrate feasibility of engineering precise nucleosome rearrangements through sequence-targeted chromatin remodeling and provide insight into targeted action and cooperation of endogenous chromatin remodelers in vivo. PMID:26993344
Hatta, M.; Liu, F.; Cirillo, L.A.
Transcriptional activity of FoxO factors is controlled through the actions of multiple growth factors signaling through protein kinase B, whereby phosphorylation of FoxO factors inhibits FoxO-mediated transactivation by promoting nuclear export. Phosphorylation of FoxO factors is enhanced by p300-mediated acetylation, which decreases their affinity for DNA. The negative effect of acetylation on FoxO DNA binding, together with nuclear FoxO mobility, is eliminated by over-expression of the de-acetylase Sirt1, suggesting that acetylation mobilizes FoxO factors in chromatin for inducible gene expression. Here, we show that acetylation significantly curtails the affinity of FoxO1 for its binding sites in nucleosomal DNA but has no effect on either stable nucleosome binding or remodeling by this factor. We suggest that, while acetylation provides a first, essential step toward mobilizing FoxO factors for inducible gene repression, additional mechanisms exist for overcoming their inherent capacity to stably bind and remodel nuclear chromatin.
van Bakel, Harm; Tsui, Kyle; Gebbia, Marinella; Mnaimneh, Sanie; Hughes, Timothy R; Nislow, Corey
Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.
van Bakel, Harm; Tsui, Kyle; Gebbia, Marinella; Mnaimneh, Sanie; Hughes, Timothy R.; Nislow, Corey
Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies. PMID:23658529
Chioda, M; Becker, P B
Nucleosome remodelling factors are regulators of DNA accessibility in chromatin and lubricators of all major functions of eukaryotic genomes. Their action is transient and reversible, yet can be decisive for irreversible cell-fate decisions during development. In addition to the well-known local actions of nucleosome remodelling factors during transcription initiation, more global and fundamental roles for remodelling complexes in shaping the epigenome during development are emerging.
Richmond, Timothy J
Nucleosomes are actively positioned along DNA by ATP-dependent, chromatin remodelling factors. A structural model for the ISW1a chromatin remodelling factor from Saccharomyces cerevisiae in complex with a dinucleosome substrate was constructed from the X-ray structures of ISW1a (ΔATPase) with and without DNA bound, two different cryo-EM (cryo-electron microscopy) structures of ISW1a (ΔATPase) bound to a nucleosome, and site-directed photo-cross-linking analyses in solution. The X-ray structure of ISW1a (ΔATPase) with DNA bound suggests that DNA sequence may be involved in nucleosome recognition and thereby specificity of promoter interaction. The model suggests how the highly ordered nucleosome arrays observed by mapping nucleosomes in genes and their promoter regions could be generated by a chromatin remodelling factor.
Lone, Imtiaz Nisar; Shukla, Manu Shubhdarshan; Charles Richard, John Lalith; Peshev, Zahary Yordanov; Dimitrov, Stefan; Angelov, Dimitar
NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A-H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.
Lone, Imtiaz Nisar; Shukla, Manu Shubhdarshan; Charles Richard, John Lalith; Peshev, Zahary Yordanov; Dimitrov, Stefan; Angelov, Dimitar
NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A–H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes. PMID:24086160
Winger, Jessica; Bowman, Gregory D
Chromatin remodelers are ATP-dependent enzymes that are critical for reorganizing and repositioning nucleosomes in concert with many basic cellular processes. For the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler, nucleosome sliding has been shown to depend on the DNA flanking the nucleosome, transcription factor binding at the nucleosome edge, and the presence of the histone H2A/H2B dimer on the entry side. Here, we report that Chd1 is also sensitive to the sequence of DNA within the nucleosome and slides nucleosomes made with the 601 Widom positioning sequence asymmetrically. Kinetic and equilibrium experiments show that poly(dA:dT) tracts perturb remodeling reactions if within one and a half helical turns of superhelix location 2 (SHL2), where the Chd1 ATPase engages nucleosomal DNA. These sequence-dependent effects do not rely on the Chd1 DNA-binding domain and are not due to differences in nucleosome affinity. Using site-specific cross-linking, we show that internal poly(dA:dT) tracts do not block the engagement of the ATPase motor with SHL2, yet they promote multiple translational positions of DNA with respect to both Chd1 and the histone core. We speculate that Chd1 senses the sequence-dependent response of DNA as the remodeler ATPase perturbs the duplex at SHL2. These results suggest that the sequence sensitivity of histones and remodelers occur at unique segments of DNA on the nucleosome, allowing them to work together or in opposition to determine nucleosome positions throughout the genome. Copyright © 2017 Elsevier Ltd. All rights reserved.
To gain deeper insights into principles of cell biology, it is essential to understand how cells reorganize their genomes by chromatin remodeling. We analyzed chromatin remodeling on next generation sequencing data from resting and activated T cells to determine a whole-genome chromatin remodeling landscape. We consider chromatin remodeling in terms of nucleosome repositioning which can be observed most robustly in long nucleosome-free regions (LNFRs) that are occupied by nucleosomes in another cell state. We found that LNFR sequences are either AT-rich or GC-rich, where nucleosome repositioning was observed much more prominently in GC-rich LNFRs — a considerable proportion of them outside promoter regions. Using support vector machines with string kernels, we identified a GC-rich DNA sequence pattern indicating loci of nucleosome repositioning in resting T cells. This pattern appears to be also typical for CpG islands. We found out that nucleosome repositioning in GC-rich LNFRs is indeed associated with CpG islands and with binding sites of the CpG-island-binding ZF-CXXC proteins KDM2A and CFP1. That this association occurs prominently inside and also prominently outside of promoter regions hints at a mechanism governing nucleosome repositioning that acts on a whole-genome scale. PMID:23144837
Mieczkowski, Jakub; Cook, April; Bowman, Sarah K.; Mueller, Britta; Alver, Burak H.; Kundu, Sharmistha; Deaton, Aimee M.; Urban, Jennifer A.; Larschan, Erica; Park, Peter J.; Kingston, Robert E.; Tolstorukov, Michael Y.
Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation. PMID:27151365
Hwang, William L; Deindl, Sebastian; Harada, Bryan T; Zhuang, Xiaowei
Imitation switch (ISWI)-family remodelling enzymes regulate access to genomic DNA by mobilizing nucleosomes. These ATP-dependent chromatin remodellers promote heterochromatin formation and transcriptional silencing by generating regularly spaced nucleosome arrays. The nucleosome-spacing activity arises from the dependence of nucleosome translocation on the length of extranucleosomal linker DNA, but the underlying mechanism remains unclear. Here we study nucleosome remodelling by human ATP-dependent chromatin assembly and remodelling factor (ACF), an ISWI enzyme comprising a catalytic subunit, Snf2h, and an accessory subunit, Acf1 (refs 2, 11 - 13). We find that ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome. Mutation of AutoN, an auto-inhibitory domain within Snf2h that bears sequence homology to the H4 tail, abolishes the linker-length sensitivity in remodelling. Addition of exogenous H4-tail peptide or deletion of the nucleosomal H4 tail also diminishes the linker-length sensitivity. Moreover, Acf1 binds both the H4-tail peptide and DNA in an amino (N)-terminal domain dependent manner, and in the ACF-bound nucleosome, lengthening the linker DNA reduces the Acf1-H4 tail proximity. Deletion of the N-terminal portion of Acf1 (or its homologue in yeast) abolishes linker-length sensitivity in remodelling and leads to severe growth defects in vivo. Taken together, our results suggest a mechanism for nucleosome spacing where linker DNA sensing by Acf1 is allosterically transmitted to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing AutoN to inhibit the ATPase activity of Snf2h. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to compete AutoN off the ATPase and thereby activating ACF.
Vandecan, Yves; Blossey, Ralf
Recent experiments have demonstrated that the ATP-utilizing chromatin assembly and remodeling factor (ACF) is a dimeric, processive motor complex which can move a nucleosome more efficiently towards longer flanking DNA than towards shorter flanking DNA strands, thereby centering an initially ill-positioned nucleosome on DNA substrates. We give a Fokker-Planck description for the repositioning process driven by transitions between internal chemical states of the remodelers. In the chemical states of ATP hydrolysis during which the repositioning takes place a power stroke is considered. The slope of the effective driving potential is directly related to ATP hydrolysis and leads to the unidirectional motion of the nucleosome-remodeler complex along the DNA strand. The Einstein force relation allows us to deduce the ATP-concentration dependence of the diffusion constant of the nucleosome-remodeler complex. We have employed our model to study the efficiency of positioning of nucleosomes as a function of the ATP sampling rate between the two motors which shows that the synchronization between the motors is crucial for the remodeling mechanism to work.
Angelov, D; Charra, M; Seve, M; Côté, J; Khochbin, S; Dimitrov, S
Here we have examined HIV-1 nucleosome remodeling upon the binding of transcription factors and the SWI/SNF complex using a novel approach. The approach combines UV laser protein-DNA crosslinking, electrophoretic mobility-shift analysis and DNase I protection analysis with immunochemical techniques. It was found that single activator-bound HIV-1 nucleosomes exhibit very weak perturbation in histone NH(2) tail-DNA interactions. However, the simultaneous binding of the transcription activators Sp1, NF-kB1, LEF-1 and USF synergistically increased the release of histone NH(2) tails from nucleosomal DNA. In contrast, the binding of SWI/SNF complex to HIV-1 nucleosome disrupted structured histone domain-DNA contacts, but not histone NH(2) tail-DNA interactions. Stable remodeled nucleosomes, (obtained after detachment of SWI/SNF), displayed identical structural alterations with those bound to SWI/SNF. These results demonstrate a different in vitro remodeling of the HIV-1 nucleosome upon the binding of multiple transcription activators and of SWI/SNF complex. Copyright 2000 Academic Press.
Grüne, Tim; Brzeski, Jan; Eberharter, Anton; Clapier, Cedric R; Corona, Davide F V; Becker, Peter B; Müller, Christoph W
Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. We have identified a substrate recognition domain in the C-terminal half of the remodeling ATPase ISWI and determined its structure by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how ISWI interacts with the nucleosomal substrate.
Montel, Fabien; Fontaine, Emeline; St-Jean, Philippe; Castelnovo, Martin; Faivre-Moskalenko, Cendrine
We propose a combined experimental (atomic force microscopy) and theoretical study of the structural and dynamical properties of nucleosomes. In contrast to biochemical approaches, this method allows us to determine simultaneously the DNA-complexed length distribution and nucleosome position in various contexts. First, we show that differences in the nucleoproteic structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce quantitative changes in the length distribution of DNA-complexed with histones. Then, the sliding action of remodeling complex SWI/SNF is characterized through the evolution of the nucleosome position and wrapped DNA length mapping. Using a linear energetic model for the distribution of DNA-complexed length, we extract the net-wrapping energy of DNA onto the histone octamer and compare it to previous studies.
Yague-Sanz, Carlo; Vázquez, Enrique; Sánchez, Mar; Antequera, Francisco; Hermand, Damien
The occupancy of nucleosomes governs access to the eukaryotic genomes and results from a combination of biophysical features and the effect of ATP-dependent remodelling complexes. Most promoter regions show a conserved pattern characterized by a nucleosome-depleted region (NDR) flanked by nucleosomal arrays. The conserved RSC remodeler was reported to be critical to establish NDR in vivo in budding yeast but other evidences suggested that this activity may not be conserved in fission yeast. By reanalysing and expanding previously published data, we propose that NDR formation requires, at least partially, RSC in both yeast species. We also discuss the most prominent biological role of RSC and the possibility that non-essential subunits do not define alternate versions of the complex.
Small, Eliza C; Xi, Liqun; Wang, Ji-Ping; Widom, Jonathan; Licht, Jonathan D
Nucleosomes, the basic unit of chromatin, have a critical role in the control of gene expression. Nucleosome positions have generally been determined by examining bulk populations of cells and then correlated with overall gene expression. Here, we describe a technique to determine nucleosome positioning in single cells by virtue of the ability of the nucleosome to protect DNA from GpC methylation. In the acid phosphatase inducible PHO5 gene, we find that there is significant cell-to-cell variation in nucleosome positions and shifts in nucleosome positioning correlate with changes in gene expression. However, nucleosome positioning is not absolute, and even with major shifts in gene expression, some cells fail to change nucleosome configuration. Mutations of the PHO5 promoter that introduce a poly(dA:dT) tract-stimulated gene expression under nonpermissive conditions led to shifts of positioned nucleosomes similar to induction of PHO5. By contrast, mutations that altered AA/TT/AT periodicity reduced gene expression upon PHO5 induction and stabilized nucleosomes in most cells, suggesting that enhanced nucleosome affinity for DNA antagonizes chromatin remodelers. Finally, we determined nucleosome positioning in two regions described as "fuzzy" or nucleosome-free when examined in a bulk assay. These regions consisted of distinct nucleosomes with a larger footprint for potential location and an increase population of cells lacking a nucleosome altogether. These data indicate an underlying complexity of nucleosome positioning that may contribute to the flexibility and heterogeneity of gene expression.
Low, Jason K K; Webb, Sarah R; Silva, Ana P G; Saathoff, Hinnerk; Ryan, Daniel P; Torrado, Mario; Brofelth, Mattias; Parker, Benjamin L; Shepherd, Nicholas E; Mackay, Joel P
Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises ∼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.
Chen, Xuefeng; Cui, Dandan; Papusha, Alma; Zhang, Xiaotian; Chu, Chia-Dwo; Tang, Jiangwu; Chen, Kaifu; Pan, Xuewen; Ira, Grzegorz
Chromosomal double-strand breaks (DSBs) are resected by 5' nucleases to form 3' single-stranded DNA substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection have been described both in cells and in vitro, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin, where histones and histone-bound proteins represent barriers for resection enzymes. Here we identify the yeast nucleosome-remodelling enzyme Fun30 as a factor promoting DSB end resection. Fun30 is the major nucleosome remodeller promoting extensive Exo1- and Sgs1-dependent resection of DSBs. The RSC and INO80 chromatin-remodelling complexes and Fun30 have redundant roles in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodelling, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads along the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5' strand processing and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of Rad9 (refs 9, 10). Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection.
Lohr, D.; Bash, R.; Wang, H.; Yodh, J.; Lindsay, S.
Atomic Force Microscopy (AFM) is a technique that can directly image single molecules in solution and it therefore provides a powerful tool for obtaining unique insights into the basic properties of biological materials and the functional processes in which they are involved. We have used AFM to analyze basic features of nucleosomes in arrays, such as DNA-histone binding strength, cooperativity in template occupation, nucleosome stabilities, nucleosome locations and the effects of acetylation, to compare these features in different types of arrays and to track the response of array nucleosomes to the action of the human Swi-Snf ATP-dependent nucleosome remodeling complex. These experiments required several specific adaptations of basic AFM methods, such as repetitive imaging of the same fields of molecules in liquid, the ability to change the environmental conditions of the sample being imaged and detection of specific types of molecules within compositionally complex samples. Here we describe the techniques that allowed such analyses to be carried out. PMID:17309844
Hainer, Sarah J; Fazzio, Thomas G
Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin-remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene-regulatory factors. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Angelov, Dimitar; Bondarenko, Vladimir A; Almagro, Sébastien; Menoni, Hervé; Mongélard, Fabien; Hans, Fabienne; Mietton, Flore; Studitsky, Vasily M; Hamiche, Ali; Dimitrov, Stefan; Bouvet, Philippe
Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A–H2B dimer, and stimulates the SWI/SNF-mediated transfer of H2A–H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription. PMID:16601700
Angelov, Dimitar; Verdel, André; An, Woojin; Bondarenko, Vladimir; Hans, Fabienne; Doyen, Cécile-Marie; Studitsky, Vassily M; Hamiche, Ali; Roeder, Robert G; Bouvet, Philippe; Dimitrov, Stefan
A histone variant H2ABbd was recently identified, but its function is totally unknown. Here we have studied the structural and functional properties of nucleosome and nucleosomal arrays reconstituted with this histone variant. We show that H2ABbd can replace the conventional H2A in the nucleosome, but this replacement results in alterations of the nucleosomal structure. The remodeling complexes SWI/SNF and ACF are unable to mobilize the variant H2ABbd nucleosome. However, SWI/SNF was able to increase restriction enzyme access to the variant nucleosome and assist the transfer of variant H2ABbd–H2B dimer to a tetrameric histone H3–H4 particle. In addition, the p300- and Gal4-VP16-activated transcription appeared to be more efficient for H2ABbd nucleosomal arrays than for conventional H2A arrays. The intriguing mechanisms by which H2ABbd affects both nucleosome remodeling and transcription are discussed. PMID:15372075
Menoni, Hervé; Di Mascio, Paolo; Cadet, Jean; Dimitrov, Stefan; Angelov, Dimitar
Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin?
Chen, Xiangmei; Shen, Yongquan; Ellis, Ronald E.
The major families of chromatin remodelers have been conserved throughout eukaryotic evolution. Because they play broad, pleiotropic roles in gene regulation, it was not known if their functions could change rapidly. Here, we show that major alterations in the use of chromatin remodelers are possible, because the nucleosome remodeling factor (NURF) complex has acquired a unique role in the sperm/oocyte decision of the nematode Caenorhabditis briggsae. First, lowering the activity of C. briggsae NURF-1 or ISW-1, the core components of the NURF complex, causes germ cells to become oocytes rather than sperm. This observation is based on the analysis of weak alleles and null mutations that were induced with TALENs and on RNA interference. Second, qRT–polymerase chain reaction data show that the C. briggsae NURF complex promotes the expression of Cbr-fog-1 and Cbr-fog-3, two genes that control the sperm/oocyte decision. This regulation occurs in the third larval stage and affects the expression of later spermatogenesis genes. Third, double mutants reveal that the NURF complex and the transcription factor TRA-1 act independently on Cbr-fog-1 and Cbr-fog-3. TRA-1 binds both promoters, and computer analyses predict that these binding sites are buried in nucleosomes, so we suggest that the NURF complex alters chromatin structure to allow TRA-1 access to Cbr-fog-1 and Cbr-fog-3. Finally, lowering NURF activity by mutation or RNA interference does not affect this trait in other nematodes, including the sister species C. nigoni, so it must have evolved recently. We conclude that altered chromatin remodeling could play an important role in evolutionary change. PMID:24987105
Chen, Xiangmei; Shen, Yongquan; Ellis, Ronald E
The major families of chromatin remodelers have been conserved throughout eukaryotic evolution. Because they play broad, pleiotropic roles in gene regulation, it was not known if their functions could change rapidly. Here, we show that major alterations in the use of chromatin remodelers are possible, because the nucleosome remodeling factor (NURF) complex has acquired a unique role in the sperm/oocyte decision of the nematode Caenorhabditis briggsae. First, lowering the activity of C. briggsae NURF-1 or ISW-1, the core components of the NURF complex, causes germ cells to become oocytes rather than sperm. This observation is based on the analysis of weak alleles and null mutations that were induced with TALENs and on RNA interference. Second, qRT-polymerase chain reaction data show that the C. briggsae NURF complex promotes the expression of Cbr-fog-1 and Cbr-fog-3, two genes that control the sperm/oocyte decision. This regulation occurs in the third larval stage and affects the expression of later spermatogenesis genes. Third, double mutants reveal that the NURF complex and the transcription factor TRA-1 act independently on Cbr-fog-1 and Cbr-fog-3. TRA-1 binds both promoters, and computer analyses predict that these binding sites are buried in nucleosomes, so we suggest that the NURF complex alters chromatin structure to allow TRA-1 access to Cbr-fog-1 and Cbr-fog-3. Finally, lowering NURF activity by mutation or RNA interference does not affect this trait in other nematodes, including the sister species C. nigoni, so it must have evolved recently. We conclude that altered chromatin remodeling could play an important role in evolutionary change. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Zhou, Weiqiang; Yan, Hong
As the fundamental repeating units in eukaryotic chromatin, nucleosomes play an important role in many biological processes. For this reason, the study of the structure of nucleosomes may help to reveal some of the crucial principals of these processes. In our research, we have used alpha shapes to model nucleosome structure and discovered that the periodic DNA dinucleotides AA, TT and GC occupy special positions in nucleosome structure with one nucleotide inside and the other outside the nucleosome surface. This structural feature and other dinucleotide characteristics can provide useful information for the study of nucleosome positioning.
Santisteban, M S; Kalashnikova, T; Smith, M M
Nucleosomes impose a block to transcription that can be overcome in vivo by remodeling complexes such as SNF/SWI and histone modification complexes such as SAGA. Mutations in the major core histones relieve transcriptional repression and bypass the requirement for SNF/SWI and SAGA. We have found that the variant histone H2A.Z regulates gene transcription, and deletion of the gene encoding H2A.Z strongly increases the requirement for SNF/SWI and SAGA. This synthetic genetic interaction is seen at the level of single genes and acts downstream of promoter nucleosome reorganization. H2A.Z is preferentially crosslinked in vivo to intergenic DNA at the PH05 and GAL1 loci, and this association changes with transcriptional activation. These results describe a novel pathway for regulating transcription using variant histones to modulate chromatin structure.
Kumar, Yatendra; Bhargava, Purnima
RNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties. Using high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at -140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes. Budding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5' NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin remodeler Isw1, aided by Isw2 and RSC
Attema, Joanne L; Reeves, Raymond; Murray, Vincent; Levichkin, Ilya; Temple, Mark D; Tremethick, David J; Shannon, M Frances
Controlled production of the cytokine IL-2 plays a key role in the mammalian immune system. Expression from the gene is tightly regulated with no detectable expression in resting T cells and a strong induction following T cell activation. The IL-2 proximal promoter (+1 to -300) contains many well-defined transcriptional activation elements that respond to T cell stimulation. To determine the role of chromatin structure in the regulation of interleukin-2 gene transcription, nucleosome assembly across the IL-2 promoter region was examined using in vitro chromatin reconstitution assays. The IL-2 promoter assembles a nucleosome that is both translationally and rotationally positioned, spanning some of the major functional control elements. The binding of transcription factors to these elements, with the exception of the architectural protein HMGA1, was occluded by the presence of the nucleosome. Analysis of the chromatin architecture of the IL-2 gene in Jurkat T cells provided evidence for the presence of a similarly positioned nucleosome in vivo. The region encompassed by this nucleosome becomes remodeled following activation of Jurkat T cells. These observations suggest that the presence of a positioned nucleosome across the IL-2 proximal promoter may play an important role in maintaining an inactive gene in resting T cells and that remodeling of this nucleosome is important for gene activation.
Zawadzki, Karl A; Morozov, Alexandre V; Broach, James R
Several well-studied promoters in yeast lose nucleosomes upon transcriptional activation and gain them upon repression, an observation that has prompted the model that transcriptional activation and repression requires nucleosome remodeling of regulated promoters. We have examined global nucleosome positioning before and after glucose-induced transcriptional reprogramming, a condition under which more than half of all yeast genes significantly change expression. The majority of induced and repressed genes exhibit no change in promoter nucleosome arrangement, although promoters that do undergo nucleosome remodeling tend to contain a TATA box. Rather, we found multiple examples where the pre-existing accessibility of putative transcription factor binding sites before glucose addition determined whether the corresponding gene would change expression in response to glucose addition. These results suggest that selection of appropriate transcription factor binding sites may be dictated to a large extent by nucleosome prepositioning but that regulation of expression through these sites is dictated not by nucleosome repositioning but by changes in transcription factor activity.
Shukla, Manu Shubhdarshan; Syed, Sajad Hussain; Goutte-Gattat, Damien; Richard, John Lalith Charles; Montel, Fabien; Hamiche, Ali; Travers, Andrew; Faivre-Moskalenko, Cendrine; Bednar, Jan; Hayes, Jeffrey J.; Angelov, Dimitar; Dimitrov, Stefan
Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a ‘defective’ docking domain may be a primary structural role of H2A.Bbd in chromatin. PMID:21131284
Shukla, Manu Shubhdarshan; Syed, Sajad Hussain; Goutte-Gattat, Damien; Richard, John Lalith Charles; Montel, Fabien; Hamiche, Ali; Travers, Andrew; Faivre-Moskalenko, Cendrine; Bednar, Jan; Hayes, Jeffrey J; Angelov, Dimitar; Dimitrov, Stefan
Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.
Nuebler, Johannes; Obermayer, Benedikt; Möbius, Wolfram; Wolff, Michael; Gerland, Ulrich
Proper positioning of nucleosomes in eukaryotic cells is important for transcription regulation. When averaged over many genes, nucleosome positions in coding regions follow a simple oscillatory pattern, which is described to a surprising degree of accuracy by a simple one-dimensional gas model for particles interacting via a soft-core repulsion. The quantitative agreement is surprising given that nucleosome positions are known to be determined by a complex interplay of mechanisms including DNA sequence-specific nucleosome stability and active repositioning of nucleosomes by remodeling enzymes. We rationalize the observed robustness of the simple oscillatory pattern by showing that the main effect of several known nucleosome positioning mechanisms is a renormalization of the particle interaction. For example, ``disorder'' from sequence-specific affinities leads to an apparent softening, while active remodeling can result in apparent softening for directional sliding or apparent stiffening for clamping mechanisms. We suggest that such parameter renormalization can explain the apparent difference of nucleosome properties in two yeast species, S. cerevisiae and S. pombe.
Wiechens, Nicola; Gkikopoulos, Triantaffyllos; Schofield, Pieta; Rocha, Sonia; Owen-Hughes, Tom
Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase’s most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements. PMID:27019336
Brzezinka, Krzysztof; Altmann, Simone; Czesnick, Hjördis; Nicolas, Philippe; Gorka, Michal; Benke, Eileen; Kabelitz, Tina; Jähne, Felix; Graf, Alexander; Kappel, Christian; Bäurle, Isabel
Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects. As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress. We show that this heat stress memory requires the activity of the FORGETTER1 (FGT1) locus, with fgt1 mutants displaying reduced maintenance of heat-induced gene expression. FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch (Sno), and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion. FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Genomic targets of the BRM remodeler overlap significantly with FGT1 targets. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1. Together, our results suggest that by modulating nucleosome occupancy, FGT1 mediates stress-induced chromatin memory. DOI: http://dx.doi.org/10.7554/eLife.17061.001 PMID:27680998
Wang, Debby D.; Yan, Hong
Nucleosomes, which contain DNA and proteins, are the basic unit of eukaryotic chromatins. Polymers such as DNA and proteins are dynamic, and their conformational changes can lead to functional changes. Periodic dinucleotide patterns exist in nucleosomal DNA chains and play an important role in the nucleosome structure. In this paper, we use normal mode analysis to detect significant structural deformations of nucleosomal DNA and investigate the relationship between periodic dinucleotides and DNA motions. We have found that periodic dinucleotides are usually located at the peaks or valleys of DNA and protein motions, revealing that they dominate the nucleosome dynamics. Also, a specific dinucleotide pattern CA/TG appears most frequently.
Chromatin remodelers perform a necessary and required function for the successful expression of our genetic code. By modifying, shifting, or ejecting nucleosomes from the chromatin structure they allow access to the underlying DNA to the rest of the cell's machinery. This research has focused on two major remodeler motors from major families of chromatin remodelers: the trimeric motor domain of RSC and the motor domain of the ISWI family, ISWI. Using primarily stopped-flow spectrofluorometry, I have categorized the time-dependent motions of these motor domains along their preferred substrate, double-stranded DNA. Combined with collected ATP utilization data, I present the subsequent analysis and associated conclusions that stem from the underlying assumptions and models. Interestingly, there is little in common between the investigated proteins aside from their favored medium. While RSC exhibits modest translocation characteristics and highly effective motion with the ability for large molecular forces, ISWI is not only structurally different but highly inefficient in its motion leading to difficulties in determining its specific translocation mechanics. While chromatin remodeling is a ubiquitous facet of eukaryotic life, there remains much to be understood about their general mechanisms.
Watanabe, Reiko; Kanno, Shin-Ichiro; Mohammadi Roushandeh, Amaneh; Ui, Ayako; Yasui, Akira
Nucleosome remodelling (NR) regulates transcription in an ATP-dependent manner, and influences gene expression required for development and cellular functions, including those involved in anti-cancer and anti-ageing processes. ATP-utilizing chromatin assembly and remodelling factor (ACF) and Brahma-associated factor (BAF) complexes, belonging to the ISWI and SWI/SNF families, respectively, are involved in various types of DNA repair. Suppression of several BAF factors makes U2OS cells significantly sensitive to X-rays, UV and especially to cisplatin, and these BAF factors contribute to the accumulation of repair proteins at various types of DNA damage and to DNA repair. Recent cancer genome sequencing and expression analysis has shown that BAF factors are frequently mutated or, more frequently, silenced in various types of cancer cells. Thus, those cancer cells are potentially X-ray- and especially cisplatin-sensitive, suggesting a way of optimizing current cancer therapy. Recent single-stem cell analysis suggests that mutations and epigenetic changes influence stem cell functionality leading to cellular ageing. Genetic and epigenetic changes in the BAF factors diminish DNA repair as well as transcriptional regulation activities, and DNA repair defects in turn negatively influence NR and transcriptional regulation. Thus, they build negative feedback loops, which accelerate both cellular senescence and transformation as common and rare cellular events, respectively, causing cellular ageing.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).
Ocampo, Josefina; Chereji, Răzvan V.; Eriksson, Peter R.; Clark, David J.
Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo. We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure. PMID:26861626
Fritzsche, W; Henderson, E
Scanning force microscopy was used to investigate the conformation of hypotonic spread chicken erythrocyte nucleosomes. Nucleosomal chains were prepared in low-salt conditions and fixed before centrifugation onto glass coverslips and air drying. The images of single nucleosomes were isolated by image processing, and the height and geometry of the resulting three-dimensional structures were investigated. An average nucleosome height of 4.2 +/- 1.1 nm was determined. A virtual cross section at half-maximum height of the nucleosome structure was used for a characterization of the nucleosome geometry. The shape of this cross section was best described by an ellipse with an aspect ratio (major/minor axis) of approximately 1.30. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:8889198
Chang, Le; Takada, Shoji
Histone tail acetylation is a key epigenetic marker that tends to open chromatin folding and activate transcription. Despite intensive studies, precise roles of individual lysine acetylation in chromatin folding have only been poorly understood. Here, we revealed structural dynamics of tri-nucleosomes with several histone tail acetylation states and analyzed histone tail interactions with DNA by performing molecular simulations at an unprecedentedly high resolution. We found versatile acetylation-dependent landscapes of tri-nucleosome. The H4 and H2A tail acetylation reduced the contact between the first and third nucleosomes mediated by the histone tails. The H3 tail acetylation reduced its interaction with neighboring linker DNAs resulting in increase of the distance between consecutive nucleosomes. Notably, two copies of the same histone in a single nucleosome have markedly asymmetric interactions with DNAs, suggesting specific pattern of nucleosome docking albeit high inherent flexibility. Estimated transcription factor accessibility was significantly high for the H4 tail acetylated structures. PMID:27698366
Duan, Ming-Rui; Smerdon, Michael J
Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.
Wu, Xueting; Liu, Hui; Liu, Hongbo; Su, Jianzhong; Lv, Jie; Cui, Ying; Wang, Fang; Zhang, Yan
Nucleosome is the elementary structural unit of eukaryotic chromatin. Instability of nucleosome positioning plays critical roles in chromatin remodeling in differentiation and disease. In this study, we investigated nucleosome dynamics in the Saccharomyces cerevisiae genome using a geometric model based on Z curve theory. We identified 52,941 stable nucleosomes and 7607 dynamic nucleosomes, compiling them into a genome-wide nucleosome dynamic positioning map and constructing a user-friendly visualization platform (http://bioinfo.hrbmu.edu.cn/nucleosome). Our approach achieved a sensitivity of 90.31% and a specificity of 87.76% for S. cerevisiae. Analysis revealed transcription factor binding sites (TFBSs) were enriched in linkers. And among the sparse nucleosomes around TFBSs, dynamic nucleosomes were slightly preferred. Gene Ontology (GO) enrichment analysis indicated that stable and dynamic nucleosomes were enriched on genes involved in different biological processes and functions. This study provides an approach for comprehending chromatin remodeling and transcriptional regulation of genes.
Kensche, Philip Reiner; Hoeijmakers, Wieteke Anna Maria; Toenhake, Christa Geeke; Bras, Maaike; Chappell, Lia; Berriman, Matthew; Bártfai, Richárd
In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.
Prochasson, Philippe; Florens, Laurence; Swanson, Selene K.; Washburn, Michael P.; Workman, Jerry L.
The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR repressor complex. The HIR complex promotes histone deposition onto DNA in vitro and constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA and nucleosomes. Furthermore, HIR complex binding to nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Thus, the HIR complex is a novel nucleosome assembly complex which functions with SWI/SNF to regulate transcription. PMID:16264190
Yadon, Adam N.; Van de Mark, Daniel; Basom, Ryan; Delrow, Jeffrey; Whitehouse, Iestyn; Tsukiyama, Toshio
Nucleosome-free regions (NFRs) at the 5′ and 3′ ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5′ and 3′ ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA. PMID:20805356
Nocetti, Nicolas; Whitehouse, Iestyn
Nucleosome repositioning at gene promoters is a fundamental aspect of the regulation of gene expression. However, the extent to which nucleosome repositioning is used within eukaryotic genomes is poorly understood. Here we report a comprehensive analysis of nucleosome positions as budding yeast transit through an ultradian cycle in which expression of >50% of all genes is highly synchronized. We present evidence of extensive nucleosome repositioning at thousands of gene promoters as genes are activated and repressed. During activation, nucleosomes are relocated to allow sites of general transcription factor binding and transcription initiation to become accessible. The extent of nucleosome shifting is closely related to the dynamic range of gene transcription and generally related to DNA sequence properties and use of the coactivators TFIID or SAGA. However, dynamic gene expression is not limited to SAGA-regulated promoters and is an inherent feature of most genes. While nucleosome repositioning occurs pervasively, we found that a class of genes required for growth experience acute nucleosome shifting as cells enter the cell cycle. Significantly, our data identify that the ATP-dependent chromatin-remodeling enzyme Snf2 plays a fundamental role in nucleosome repositioning and the expression of growth genes. We also reveal that nucleosome organization changes extensively in concert with phases of the cell cycle, with large, regularly spaced nucleosome arrays being established in mitosis. Collectively, our data and analysis provide a framework for understanding nucleosome dynamics in relation to fundamental DNA-dependent transactions.
Sun, Jian; Zhang, Qing; Schlick, Tamar
Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended "beads-on-a-string" conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA-nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt. chromatin modeling | irregular 3D zig-zag | Discrete Surface Charge Optimization model
Sun, Jian; Zhang, Qing; Schlick, Tamar
Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended “beads-on-a-string” conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA–nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt. PMID:15919827
Lesbats, Paul; Botbol, Yair; Chevereau, Guillaume; Vaillant, Cédric; Calmels, Christina; Arneodo, Alain; Andreola, Marie-Line; Lavigne, Marc; Parissi, Vincent
Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier. PMID:21347347
Hepp, Matias I.; Alarcon, Valentina; Dutta, Arnob; Workman, Jerry L.; Gutiérrez, José L.
Regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key actors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues. PMID:24972368
Raj, Ritu; Lercher, Lukas; Mohammed, Shabaz
Abstract Transcriptional regulation can be established by various post‐translational modifications (PTMs) on histone proteins in the nucleosome and by nucleobase modifications on chromosomal DNA. Functional consequences of histone O‐GlcNAcylation (O‐GlcNAc=O‐linked β‐N‐acetylglucosamine) are largely unexplored. Herein, we generate homogeneously GlcNAcylated histones and nucleosomes by chemical post‐translational modification. Mass‐spectrometry‐based quantitative interaction proteomics reveals a direct interaction between GlcNAcylated nucleosomes and the “facilitates chromatin transcription” (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes may point towards O‐GlcNAcylation as one of the triggers for FACT‐driven transcriptional control. PMID:27272618
Ramachandran, Srinivas; Zentner, Gabriel E; Henikoff, Steven
Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.
Visnapuu, Mari-Liis; Greene, Eric C.
Here we use single-molecule imaging to determine coarse-grained intrinsic energy landscapes for nucleosome deposition on model DNA substrates. Our results reveal distributions that are correlated with recent in silico predictions, reinforcing the hypothesis that DNA contains some intrinsic positioning information. We also show that cis-regulatory sequences in human DNA coincide with peaks in the intrinsic landscape, whereas valleys correspond to non-regulatory regions, and we present evidence arguing that nucleosome deposition in vertebrates is influenced by factors not accounted for by current theory. Finally, we demonstrate that intrinsic landscapes of nucleosomes containing the centromere-specific variant CenH3 are correlated with patterns observed for canonical nucleosomes, arguing that CenH3 does not alter sequence preferences of centromeric nucleosomes. However, the non-histone protein Scm3 alters the intrinsic landscape of CenH3-containing nucleosomes, enabling them to overcome the otherwise exclusionary effects of poly(dA–dT) tracts, which are enriched in centromeric DNA. PMID:19734899
Lieleg, Corinna; Krietenstein, Nils; Walker, Maria; Korber, Philipp
Eukaryotic nuclear DNA is packaged into nucleosomes. During the past decade, genome-wide nucleosome mapping across species revealed the high degree of order in nucleosome positioning. There is a conserved stereotypical nucleosome organization around transcription start sites (TSSs) with a nucleosome-depleted region (NDR) upstream of the TSS and a TSS-aligned regular array of evenly spaced nucleosomes downstream over the gene body. As nucleosomes largely impede access to DNA and thereby provide an important level of genome regulation, it is of general interest to understand the mechanisms generating nucleosome positioning and especially the stereotypical NDR-array pattern. We focus here on the most advanced models, unicellular yeasts, and review the progress in mapping nucleosomes and which nucleosome positioning mechanisms are discussed. There are four mechanistic aspects: How are NDRs generated? How are individual nucleosomes positioned, especially those flanking the NDRs? How are nucleosomes evenly spaced leading to regular arrays? How are regular arrays aligned at TSSs? The main candidates for nucleosome positioning determinants are intrinsic DNA binding preferences of the histone octamer, specific DNA binding factors, nucleosome remodeling enzymes, transcription, and statistical positioning. We summarize the state of the art in an integrative model where nucleosomes are positioned by a combination of all these candidate determinants. We highlight the predominance of active mechanisms involving nucleosome remodeling enzymes which may be recruited by DNA binding factors and the transcription machinery. While this mechanistic framework emerged clearly during recent years, the involved factors and their mechanisms are still poorly understood and require future efforts combining in vivo and in vitro approaches.
Ringel, Alison E.; Cieniewicz, Anne M.; Taverna, Sean D.; Wolberger, Cynthia
The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications. PMID:26401015
Ye, Zhenqing; Chen, Zhong; Sunkel, Benjamin; Frietze, Seth; Huang, Tim H.-M.; Wang, Qianben; Jin, Victor X.
The compaction of nucleosomal structures creates a barrier for DNA-binding transcription factors (TFs) to access their cognate cis-regulatory elements. Pioneer factors (PFs) such as FOXA1 are able to directly access these cis-targets within compact chromatin. However, how these PFs interplay with nucleosomes remains to be elucidated, and is critical for us to understand the underlying mechanism of gene regulation. Here, we have conducted a computational analysis on a strand-specific paired-end ChIP-exo (termed as ChIP-ePENS) data of FOXA1 in LNCaP cells by our novel algorithm ePEST. We find that FOXA1 chromatin binding occurs via four distinct border modes (or footprint boundary patterns), with a preferential footprint boundary patterns relative to FOXA1 motif orientation. In addition, from this analysis three fundamental nucleotide positions (oG, oS and oH) emerged as major determinants for blocking exo-digestion and forming these four distinct border modes. By integrating histone MNase-seq data, we found an astonishingly consistent, ‘well-positioned’ configuration occurs between FOXA1 motifs and dyads of nucleosomes genome-wide. We further performed ChIP-seq of eight chromatin remodelers and found an increased occupancy of these remodelers on FOXA1 motifs for all four border modes (or footprint boundary patterns), indicating the full occupancy of FOXA1 complex on the three blocking sites (oG, oS and oH) likely produces an active regulatory status with well-positioned phasing for protein binding events. Together, our results suggest a positional-nucleosome-oriented accessing model for PFs seeking target motifs, in which FOXA1 can examine each underlying DNA nucleotide and is able to sense all potential motifs regardless of whether they face inward or outward from histone octamers along the DNA helix axis. PMID:27458208
Wei, Sijie; Falk, Samantha J.; Black, Ben E.; Lee, Tae-Hee
Structural dynamics of nucleic acid and protein is an important physical basis of their functions. These motions are often very difficult to synchronize and too fast to be clearly resolved with the currently available single molecule methods. Here we demonstrate a novel hybrid single molecule approach combining stochastic data analysis with fluorescence correlation that enables investigations of sub-ms unsynchronized structural dynamics of macromolecules. Based on the method, we report the first direct evidence of spontaneous DNA motions at the nucleosome termini. The nucleosome, comprising DNA and a histone core, is the fundamental packing unit of eukaryotic genes that must be accessed during various genome transactions. Spontaneous DNA opening at the nucleosome termini has long been hypothesized to enable gene access in the nucleosome, but has yet to be directly observed. Our approach reveals that DNA termini in the nucleosome open and close repeatedly at 0.1–1 ms−1. The kinetics depends on salt concentration and DNA–histone interactions but not much on DNA sequence, suggesting that this dynamics is universal and imposes the kinetic limit to gene access. These results clearly demonstrate that our method provides an efficient and robust means to investigate unsynchronized structural changes of DNA at a sub-ms time resolution. PMID:26013809
Bantele, Susanne CS; Ferreira, Pedro; Gritenaite, Dalia; Boos, Dominik; Pfander, Boris
DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here, we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Budding yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11/TOPBP1, respectively. In yeast, this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin, and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice. DOI: http://dx.doi.org/10.7554/eLife.21687.001 PMID:28063255
Cherry, Christopher M; Matunis, Erika L
Regulation of stem cells depends on both tissue-specific transcriptional regulators and changes in chromatin organization, yet the coordination of these events in endogenous niches is poorly understood. In the Drosophila testis, local JAK-STAT signaling maintains germline and somatic stem cells (GSCs and cyst progenitor cells, or CPCs) in a single niche. Here we show that epigenetic regulation via the nucleosome-remodeling factor (NURF) complex ensures GSC and CPC maintenance by positively regulating JAK-STAT signaling, thereby preventing premature differentiation. Conversely, NURF is not required in early differentiating daughter cells of either lineage. Because three additional ATP-dependent chromatin remodelers (ACF, CHRAC, and dMi-2/NuRD) are dispensable for stem cell maintenance in the testis, epigenetic regulation of stem cells within this niche may rely primarily on NURF. Thus, local signals cooperate with specific chromatin-remodeling complexes in intact niches to coordinately regulate a common set of target genes to prevent premature stem cell differentiation.
Chen, Kaifu; Wilson, Marenda A; Hirsch, Calley; Watson, Anjanette; Liang, Shoudan; Lu, Yue; Li, Wei; Dent, Sharon Y R
The yeast Cyc8 (also known as Ssn6)-Tup1 complex regulates gene expression through a variety of mechanisms, including positioning of nucleosomes over promoters of some target genes to limit accessibility to the transcription machinery. To further define the functions of Cyc8-Tup1 in gene regulation and chromatin remodeling, we performed genome-wide profiling of changes in nucleosome organization and gene expression that occur upon loss of CYC8 or TUP1 and observed extensive nucleosome alterations in both promoters and gene bodies of derepressed genes. Our improved nucleosome profiling and analysis approaches revealed low-occupancy promoter nucleosomes (P nucleosomes) at locations previously defined as nucleosome-free regions. In the absence of CYC8 or TUP1, this P nucleosome is frequently lost, whereas nucleosomes are gained at -1 and +1 positions, accompanying up-regulation of downstream genes. Our analysis of public ChIP-seq data revealed that Cyc8 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of CYC8 or TUP1. These results suggest that stabilization of the P nucleosome on TATA-containing promoters may be a central feature of the repressive chromatin architecture created by the Cyc8-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.
Guryanova, Olga A.; Shank, Kaitlyn; Spitzer, Barbara; Luciani, Luisa; Koche, Richard P.; Garrett-Bakelman, Francine E.; Ganzel, Chezi; Durham, Benjamin H.; Mohanty, Abhinita; Hoermann, Gregor; Rivera, Sharon A.; Chramiec, Alan G.; Pronier, Elodie; Bastian, Lennart; Keller, Matthew D.; Tovbin, Daniel; Loizou, Evangelia; Weinstein, Abby R.; Gonzalez, Adriana Rodriguez; Lieu, Yen; Rowe, Jacob M.; Pastore, Friederike; McKenney, Anna Sophia; Krivtsov, Andrei V.; Sperr, Wolfgang R.; Cross, Justin R.; Mason, Christopher E.; Tallman, Martin S.; Arcila, Maria E.; Abdel-Wahab, Omar; Armstrong, Scott A.; Kubicek, Stefan; Staber, Philipp B.; Gönen, Mithat; Paietta, Elisabeth M.; Melnick, Ari M.; Nimer, Stephen D.; Mukherjee, Siddhartha; Levine, Ross L.
Although the majority of acute myeloid leukemia (AML) patients initially respond to chemotherapy, many patients subsequently relapse; the mechanistic basis for AML persistence following chemotherapy has not been delineated. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3Amut), are observed in AML1–3 and in individuals with clonal hematopoiesis in the absence of leukemic transformation4,5. DNMT3Amut AML patients have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy6,7, suggesting that DNMT3Amut cells persist and drive relapse8. Here we show that Dnmt3amut induces hematopoietic stem cell (HSC) expansion, cooperates with Flt3ITD and Npm1c to induce AML in vivo, and promotes resistance to anthracycline chemotherapy. In AML patients, DNMT3AR882 mutations predict for minimal residual disease (MRD), underscoring their role in AML chemoresistance. DNMT3Amut cells show impaired nucleosome eviction and chromatin remodeling in response to anthracyclines, resulting from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect leads to an inability to sense and repair DNA torsional stress, which results in increased mutagenesis. Our studies identify a critical role for DNMT3AR882 mutations in driving AML chemoresistance, and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy. PMID:27841873
Deindl, Sebastian; Zhuang, Xiaowei
Due to its ability to track distance changes within individual molecules or molecular complexes on the nanometer scale and in real time, single-molecule fluorescence resonance energy transfer (single-molecule FRET) is a powerful tool to tackle a wide range of important biological questions. Using our recently developed single-molecule FRET assay to monitor nucleosome translocation as an illustrative example, we describe here in detail how to set up, carry out, and analyze single-molecule FRET experiments that provide time-dependent information on biomolecular processes. PMID:22929765
Börner, Kenneth; Jain, Dhawal; Vazquez-Pianzola, Paula; Vengadasalam, Sandra; Steffen, Natascha; Fyodorov, Dmitry V; Tomancak, Pavel; Konev, Alexander; Suter, Beat; Becker, Peter B
The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF1 subunit and a pair of small histone-like proteins, CHRAC-14/16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. Expression of the signature subunit ACF1 is restricted during embryonic development, but remains high in primordial germ cells. Therefore, we explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf1(7) allele leads to defective egg chambers and their elimination through apoptosis. In addition, we find a variety of unusual 16-cell cyst packaging phenotypes in the previously known Acf1(1) allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, we found that the Acf1(1) deletion--despite disruption of the Acf1 reading frame--expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis.
Börner, Kenneth; Jain, Dhawal; Vazquez-Pianzola, Paula; Vengadasalam, Sandra; Steffen, Natascha; Fyodorov, Dmitry V.; Tomancak, Pavel; Konev, Alexander; Suter, Beat; Becker, Peter B.
The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF1 subunit and a pair of small histone-like proteins, CHRAC-14/16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. Expression of the signature subunit ACF1 is restricted during embryonic development, but remains high in primordial germ cells. Therefore, we explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf17 allele leads to defective egg chambers and their elimination through apoptosis. In addition, we find a variety of unusual 16-cell cyst packaging phenotypes in the previously known Acf11 allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, we found that the Acf11 deletion – despite disruption of the Acf1 reading frame – expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis. PMID:26851213
Chereji, Răzvan V; Morozov, Alexandre V
Nucleosomes may undergo a conformational change in which a stretch of DNA peels off the histone octamer surface as a result of thermal fluctuations or interactions with chromatin remodelers. Thus, neighboring nucleosomes may invade each other's territories by DNA unwrapping and translocation, or through initial assembly in partially wrapped states. A recent high-resolution map of distances between dyads of neighboring nucleosomes in Saccharomyces cerevisiae reveals that nucleosomes frequently overlap DNA territories of their neighbors. This conclusion is supported by lower-resolution maps of S. cerevisiae nucleosome lengths based on micrococcal nuclease digestion and paired-end sequencing. The average length of wrapped DNA follows a stereotypical pattern in genes and promoters, correlated with the well-known distribution of nucleosome occupancy: nucleosomal DNA tends to be shorter in promoters and longer in coding regions. To explain these observations, we have developed a biophysical model that uses a 10-11-bp periodic histone-DNA binding energy profile. The profile is based on the pattern of histone-DNA contacts in nucleosome crystal structures, as well as the idea of linker length discretization caused by higher-order chromatin structure. Our model is in agreement with the observed genome-wide distributions of interdyad distances, wrapped DNA lengths, and nucleosome occupancies. Furthermore, our approach explains in vitro measurements of the accessibility of nucleosome-covered target sites and nucleosome-induced cooperativity between DNA-binding factors. We rule out several alternative scenarios of histone-DNA interactions as inconsistent with the genomic data.
Yoo, Miran; Choi, Kwang-Yeon; Kim, Jieun; Kim, Mujun; Shim, Jaehoon; Choi, Jun-Hyeok; Cho, Hye-Yeon; Oh, Jung-Pyo; Kim, Hyung-Su; Kaang, Bong-Kiun; Han, Jin-Hee
Although epigenetic mechanisms of gene expression regulation have recently been implicated in memory consolidation and persistence, the role of nucleosome-remodeling is largely unexplored. Recent studies show that the functional loss of BAF53b, a postmitotic neuron-specific subunit of the BAF nucleosome-remodeling complex, results in the deficit of consolidation of hippocampus-dependent memory and cocaine-associated memory in the rodent brain. However, it is unclear whether BAF53b expression is regulated during memory formation and how BAF53b regulates fear memory in the amygdala, a key brain site for fear memory encoding and storage. To address these questions, we used viral vector approaches to either decrease or increase BAF53b function specifically in the lateral amygdala of adult mice in auditory fear conditioning paradigm. Knockdown of Baf53b before training disrupted long-term memory formation with no effect on short-term memory, basal synaptic transmission, and spine structures. We observed in our qPCR analysis that BAF53b was induced in the lateral amygdala neurons at the late consolidation phase after fear conditioning. Moreover, transient BAF53b overexpression led to persistently enhanced memory formation, which was accompanied by increase in thin-type spine density. Together, our results provide the evidence that BAF53b is induced after learning, and show that such increase of BAF53b level facilitates memory consolidation likely by regulating learning-related spine structural plasticity.SIGNIFICANCE STATEMENT Recent works in the rodent brain begin to link nucleosome remodeling-dependent epigenetic mechanism to memory consolidation. Here we show that BAF53b, an epigenetic factor involved in nucleosome remodeling, is induced in the lateral amygdala neurons at the late phase of consolidation after fear conditioning. Using specific gene knockdown or overexpression approaches, we identify the critical role of BAF53b in the lateral amygdala neurons for memory
Miller, Anzy; Ralser, Meryem; Kloet, Susan L.; Loos, Remco; Nishinakamura, Ryuichi; Bertone, Paul; Vermeulen, Michiel
Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, we show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. We propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells. PMID:27471257
Nikalayevich, Elvira; Ohkura, Hiroyuki
ABSTRACT Chromosome condensation during cell division is one of the most dramatic events in the cell cycle. Condensin and topoisomerase II are the most studied factors in chromosome condensation. However, their inactivation leads to only mild defects and little is known about the roles of other factors. Here, we took advantage of Drosophila oocytes to elucidate the roles of potential condensation factors by performing RNA interference (RNAi). Consistent with previous studies, depletion of condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, we found severe undercondensation of chromosomes after depletion of the Mi-2-containing NuRD nucleosome remodelling complex or the protein kinase NHK-1 (also known as Ballchen in Drosophila). The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways of chromosome condensation. We show that the main role of NHK-1 in chromosome condensation is to phosphorylate Barrier-to-autointegration factor (BAF) and suppress its activity in linking chromosomes to nuclear envelope proteins. We further show that NHK-1 is important for chromosome condensation during mitosis as well as in oocytes. PMID:25501812
Nikalayevich, Elvira; Ohkura, Hiroyuki
Chromosome condensation during cell division is one of the most dramatic events in the cell cycle. Condensin and topoisomerase II are the most studied factors in chromosome condensation. However, their inactivation leads to only mild defects and little is known about the roles of other factors. Here, we took advantage of Drosophilaoocytes to elucidate the roles of potential condensation factors by performing RNA interference (RNAi). Consistent with previous studies, depletion of condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, we found severe undercondensation of chromosomes after depletion of the Mi-2-containing NuRD nucleosome remodelling complex or the protein kinase NHK-1 (also known as Ballchen in Drosophila). The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways of chromosome condensation. We show that the main role of NHK-1 in chromosome condensation is to phosphorylate Barrier-to-autointegration factor (BAF) and suppress its activity in linking chromosomes to nuclear envelope proteins. We further show that NHK-1 is important for chromosome condensation during mitosis as well as in oocytes.
Becker, Peter B
Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.
Xie, Wenbing; Ling, Te; Zhou, Yonggang; Feng, Weijun; Zhu, Qiaoyun; Stunnenberg, Henk G; Grummt, Ingrid; Tao, Wei
rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.
Moyle-Heyrman, Georgette; Zaichuk, Tetiana; Xi, Liqun; Zhang, Quanwei; Uhlenbeck, Olke C.; Holmgren, Robert; Widom, Jonathan; Wang, Ji-Ping
Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the −1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content. PMID:24277842
Moyle-Heyrman, Georgette; Zaichuk, Tetiana; Xi, Liqun; Zhang, Quanwei; Uhlenbeck, Olke C; Holmgren, Robert; Widom, Jonathan; Wang, Ji-Ping
Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the -1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content.
Segal, Eran; Widom, Jonathan
The DNA of eukaryotic genomes is wrapped in nucleosomes, which strongly distort and occlude the DNA from access to most DNA-binding proteins. An understanding of the mechanisms that control nucleosome positioning along the DNA is thus essential to understanding the binding and action of proteins that carry out essential genetic functions. New genome-wide data on in vivo and in vitro nucleosome positioning greatly advance our understanding of several factors that can influence nucleosome positioning, including DNA sequence preferences, DNA methylation, histone variants and post-translational modifications, higher order chromatin structure, and the actions of transcription factors, chromatin remodelers and other DNA-binding proteins. We discuss how these factors function and ways in which they might be integrated into a unified framework that accounts for both the preservation of nucleosome positioning and the dynamic nucleosome repositioning that occur across biological conditions, cell types, developmental processes and disease.
Brower-Toland, Brent D.; Smith, Corey L.; Yeh, Richard C.; Lis, John T.; Peterson, Craig L.; Wang, Michelle D.
The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.
Ha, Misook; Kraushaar, Daniel C; Zhao, Keji
The histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification. H3.3 is deposited by various chaperones at regulatory sites, gene bodies, and certain heterochromatic sites such as telomeres and centromeres. Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers, and epigenetic marks. Our comprehensive analysis of H3.3 dissociation rates revealed distinct H3.3 dissociation dynamics at various functional chromatin domains. At transcription start sites, H3.3 dissociates rapidly with the highest rate at nucleosome-depleted regions (NDRs) just upstream of Pol II binding, followed by low H3.3 dissociation rates across gene bodies. H3.3 turnover at transcription start sites, gene bodies, and transcription end sites was positively correlated with transcriptional activity. H3.3 is found decorated with various histone modifications that regulate transcription and maintain chromatin integrity. We find greatly varying H3.3 dissociation rates across various histone modification domains: high dissociation rates at active histone marks and low dissociation rates at heterochromatic marks. Well- defined zones of high H3.3-nucleosome turnover were detected at binding sites of ESC-specific pluripotency factors and chromatin remodelers, suggesting an important role for H3.3 in facilitating protein binding. Among transcription factor binding sites we detected higher H3.3 turnover at distal cis-acting sites compared to proximal genic transcription factor binding sites. Our results imply that fast H3.3 dissociation is a hallmark of interactions between DNA and transcriptional regulators. Our study demonstrates that H3.3 turnover and
Pass, Daniel Antony; Sornay, Emily; Marchbank, Angela; Crawford, Margaret R; Paszkiewicz, Konrad; Kent, Nicholas A; Murray, James A H
All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more
Qiu, Hongfang; Chereji, Răzvan V.; Hu, Cuihua; Cole, Hope A.; Rawal, Yashpal; Clark, David J.; Hinnebusch, Alan G.
Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes. PMID:26602697
Valieva, Maria E.; Gerasimova, Nadezhda S.; Kudryashova, Kseniya S.; Kozlova, Anastasia L.; Kirpichnikov, Mikhail P.; Hu, Qi; Botuyan, Maria Victoria; Mer, Georges; Feofanov, Alexey V.; Studitsky, Vasily M.
A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar. PMID:28067802
Wakamori, Masatoshi; Fujii, Yoshifumi; Suka, Noriyuki; Shirouzu, Mikako; Sakamoto, Kensaku; Umehara, Takashi; Yokoyama, Shigeyuki
Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs. PMID:26607036
Weisbrod, S T
Nucleosomes from actively transcribed genes (active nucleosomes) contain nonhistone proteins HMG 14 and 17 and are preferentially sensitive to digestion by DNAse I. Active nucleosomes isolated by chromatography on an HMG 14 and 17 glass bead affinity column were analyzed with respect to overall structure, accessory nonhistone components and modifications to the DNA and histones. The experiments lead to the following conclusions: the DNA in the active nucleosome is undermethylated compared to bulk DNA; topoisomerase I is a non-stoichiometric component of the active nucleosome fraction; the level of histone acetylation is enriched in active nucleosomes, but the extent of enrichment cannot account for HMG binding; and the two histone H3 molecules in the active nucleosome can dimerize more readily and are, therefore, probably closer together than those in the bulk of the nucleosomes. Additionally it is shown that HMG 14 and 17 prefer to bind to single- vs. double-stranded nucleic acids. The role of HMG 14 and 17 in producing a highly DNAse I sensitive structure and correspondingly helping to facilitate transcription is discussed in terms of these properties. Images PMID:6210882
Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; Martínez-Fernández, Verónica; Fernández-Pevida, Antonio; Cuevas-Bermúdez, Abel; Martín-Expósito, Manuel; Chávez, Sebastián; de la Cruz, Jesús; Navarro, Francisco
Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity. PMID:25081216
Freaney, Jonathan E; Zhang, Quanwei; Yigit, Erbay; Kim, Rebecca; Widom, Jonathan; Wang, Ji-Ping; Horvath, Curt M
Genome-wide investigations have dramatically increased our understanding of nucleosome positioning and the role of chromatin in gene regulation, yet some genomic regions have been poorly represented in human nucleosome maps. One such region is represented by human chromosome 9p21-22, which contains the type I interferon gene cluster that includes 16 interferon alpha genes and the single interferon beta, interferon epsilon, and interferon omega genes. A high-density nucleosome mapping strategy was used to generate locus-wide maps of the nucleosome organization of this biomedically important locus at a steady state and during a time course of infection with Sendai virus, an inducer of interferon gene expression. Detailed statistical and computational analysis illustrates that nucleosomes in this locus exhibit preferences for particular dinucleotide and oligomer DNA sequence motifs in vivo, which are similar to those reported for lower eukaryotic nucleosome-DNA interactions. These data were used to visualize the region's chromatin architecture and reveal features that are common to the organization of all the type I interferon genes, indicating a common nucleosome-mediated gene regulatory paradigm. Additionally, this study clarifies aspects of the dynamic changes that occur with the nucleosome occupying the transcriptional start site of the interferon beta gene after virus infection.
Schwab, David J.; Bruinsma, Robijn F.; Rudnick, Joseph; Widom, Jonathan
We present a statistical-mechanical model for the positioning of nucleosomes along genomic DNA molecules as a function of the strength of the binding potential and the chemical potential of the nucleosomes. We show that a significant section of the DNA is composed of two-level nucleosome switching regions where the nucleosome distribution undergoes a localized, first-order transition. The location of the nucleosome switches shows a strong correlation with the location of gene-regulation regions.
Schwab, David J; Bruinsma, Robijn F; Rudnick, Joseph; Widom, Jonathan
We present a statistical-mechanical model for the positioning of nucleosomes along genomic DNA molecules as a function of the strength of the binding potential and the chemical potential of the nucleosomes. We show that a significant section of the DNA is composed of two-level nucleosome switching regions where the nucleosome distribution undergoes a localized, first-order transition. The location of the nucleosome switches shows a strong correlation with the location of gene-regulation regions.
Kelly, Theresa K.; Miranda, Tina Branscombe; Liang, Gangning; Berman, Benjamin P.; Lin, Joy C.; Tanay, Amos; Jones, Peter A.
Profound chromatin changes occur during mitosis to allow for gene silencing and chromosome segregation followed by re-activation of memorized transcription states in daughter cells. Using genome-wide sequencing, we found H2A.Z containing +1 nucleosomes of active genes shift upstream to occupy TSSs during mitosis, significantly reducing nucleosome-depleted regions. Single molecule analysis confirmed nucleosome shifting and demonstrated that mitotic shifting is specific to active genes that are silenced during mitosis and thus is not seen on promoters, which are silenced by methylation or mitotically expressed genes. Using the GRP78 promoter as a model, we found H3K4 tri-methylation is also maintained while other indicators of active chromatin are lost and expression is decreased. These key changes provide a potential mechanism for rapid silencing and re-activation of genes during the cell cycle. PMID:20864037
Tsankov, Alex; Yanagisawa, Yoshimi; Rhind, Nicholas; Regev, Aviv; Rando, Oliver J.
The packaging of eukaryotic genomes into nuclesomes plays critical roles in chromatin organization and gene regulation. Studies in Saccharomyces cerevisiae indicate that nucleosome occupancy is partially encoded by intrinsic antinucleosomal DNA sequences, such as poly(A) sequences, as well as by binding sites for trans-acting factors that can evict nucleosomes, such as Reb1 and the Rsc3/30 complex. Here, we use genome-wide nucleosome occupancy maps in 13 Ascomycota fungi to discover large-scale evolutionary reprogramming of both intrinsic and trans determinants of chromatin structure. We find that poly(G)s act as intrinsic antinucleosomal sequences, comparable to the known function of poly(A)s, but that the abundance of poly(G)s has diverged greatly between species, obscuring their antinucleosomal effect in low-poly(G) species such as S. cerevisiae. We also develop a computational method that uses nucleosome occupancy maps for discovering trans-acting general regulatory factor (GRF) binding sites. Our approach reveals that the specific sequences bound by GRFs have diverged substantially across evolution, corresponding to a number of major evolutionary transitions in the repertoire of GRFs. We experimentally validate a proposed evolutionary transition from Cbf1 as a major GRF in pre-whole-genome duplication (WGD) yeasts to Reb1 in post-WGD yeasts. We further show that the mating type switch-activating protein Sap1 is a GRF in S. pombe, demonstrating the general applicability of our approach. Our results reveal that the underlying mechanisms that determine in vivo chromatin organization have diverged and that comparative genomics can help discover new determinants of chromatin organization. PMID:21914852
Hauk, G.; McKnight, J; Nodelman, I; Bowman, G
Chromatin remodelers are ATP-driven machines that assemble, slide, and remove nucleosomes from DNA, but how the ATPase motors of remodelers are regulated is poorly understood. Here we show that the double chromodomain unit of the Chd1 remodeler blocks DNA binding and activation of the ATPase motor in the absence of nucleosome substrates. The Chd1 crystal structure reveals that an acidic helix joining the chromodomains can pack against a DNA-binding surface of the ATPase motor. Disruption of the chromodomain-ATPase interface prevents discrimination between nucleosomes and naked DNA and reduces the reliance on the histone H4 tail for nucleosome sliding. We propose that the chromodomains allow Chd1 to distinguish between nucleosomes and naked DNA by physically gating access to the ATPase motor, and we hypothesize that related ATPase motors may employ a similar strategy to discriminate among DNA-containing substrates.
Dechassa, Mekonnen Lemma; Sabri, Abdellah; Pondugula, Santhi; Kassabov, Stefan R; Chatterjee, Nilanjana; Kladde, Michael P; Bartholomew, Blaine
The ATP-dependent chromatin remodeling complex SWI/SNF regulates transcription and has been implicated in promoter nucleosome eviction. Efficient nucleosome disassembly by SWI/SNF alone in biochemical assays, however, has not been directly observed. Employing a model system of dinucleosomes rather than mononucleosomes, we demonstrate that remodeling leads to ordered and efficient disassembly of one of the two nucleosomes. An H2A/H2B dimer is first rapidly displaced, and then, in a slower reaction, an entire histone octamer is lost. Nucleosome disassembly by SWI/SNF did not require additional factors such as chaperones or acceptors of histones. Observations in single molecules as well as bulk measurement suggest that a key intermediate in this process is one in which a nucleosome is moved toward the adjacent nucleosome. SWI/SNF recruited by the transcriptional activator Gal4-VP16 preferentially mobilizes the proximal nucleosome and destabilizes the adjacent nucleosome.
Chereji, Răzvan V; Morozov, Alexandre V
Nucleosome is a histone-DNA complex known as the fundamental repeating unit of chromatin. Up to 90% of eukaryotic DNA is wrapped around consecutive octamers made of the core histones H2A, H2B, H3 and H4. Nucleosome positioning affects numerous cellular processes that require robust and timely access to genomic DNA, which is packaged into the tight confines of the cell nucleus. In living cells, nucleosome positions are determined by intrinsic histone-DNA sequence preferences, competition between histones and other DNA-binding proteins for genomic sequence, and ATP-dependent chromatin remodelers. We discuss the major energetic contributions to nucleosome formation and remodeling, focusing especially on partial DNA unwrapping off the histone octamer surface. DNA unwrapping enables efficient access to nucleosome-buried binding sites and mediates rapid nucleosome removal through concerted action of two or more DNA-binding factors. High-resolution, genome-scale maps of distances between neighboring nucleosomes have shown that DNA unwrapping and nucleosome crowding (mutual invasion of nucleosome territories) are much more common than previously thought. Ultimately, constraints imposed by nucleosome energetics on the rates of ATP-dependent and spontaneous chromatin remodeling determine nucleosome occupancy genome-wide, and shape pathways of cellular response to environmental stresses.
Trifonov, Edward N; Nibhani, Reshma
Don Crothers, Mikael Kubista, Jon Widom, and their teams have been first to look for strong nucleosomes, in a bid to reveal the nucleosome positioning pattern(s) carried by the nucleosome DNA sequences. They were first to demonstrate that the nucleosome stability correlates with 10-11 base sequence periodicity, and that the strong nucleosomes localize preferentially in centromeres. This review describes these findings and their connection to recent discovery of the strong nucleosomes (SNs) with visibly periodic nucleosome DNA sequences.
Volkman, Loy E.
Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.
Kawashima, Satoshi; Nakabayashi, Yu; Matsubara, Kazuko; Sano, Norihiko; Enomoto, Takemi; Tanaka, Kozo; Seki, Masayuki; Horikoshi, Masami
The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere-specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule-depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty-three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS-I, -II, and -III (TBZ/benomyl-sensitive regions I–III). Elevation of mono-polar attachment induced by prior nocodazole treatment was observed in H2A-I112A (TBS-I), H2A-E57A (TBS-II), and H4-L97A (TBS-III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi-orientation, occurred in H2A-I112A and H2A-E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4-L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS-I and -II, are necessary for Sgo1-mediated chromosome bi-orientation and that TBS-III is required for Htz1 function. PMID:21772248
Jansen, An; Verstrepen, Kevin J.
Summary: The DNA of eukaryotic cells is spooled around large histone protein complexes, forming nucleosomes that make up the basis for a high-order packaging structure called chromatin. Compared to naked DNA, nucleosomal DNA is less accessible to regulatory proteins and regulatory processes. The exact positions of nucleosomes therefore influence several cellular processes, including gene expression, chromosome segregation, recombination, replication, and DNA repair. Here, we review recent technological advances enabling the genome-wide mapping of nucleosome positions in the model eukaryote Saccharomyces cerevisiae. We discuss the various parameters that determine nucleosome positioning in vivo, including cis factors like AT content, variable tandem repeats, and poly(dA:dT) tracts that function as chromatin barriers and trans factors such as chromatin remodeling complexes, transcription factors, histone-modifying enzymes, and RNA polymerases. In the last section, we review the biological role of chromatin in gene transcription, the evolution of gene regulation, and epigenetic phenomena. PMID:21646431
Moyle-Heyrman, Georgette; Tims, Hannah S; Widom, Jonathan
Cooperativity in transcription factor (TF) binding is essential in eukaryotic gene regulation and arises through diverse mechanisms. Here, we focus on one mechanism, collaborative competition, which is of interest because it arises both automatically (with no requirement for TF coevolution) and spontaneously (with no requirement for ATP-dependent nucleosome remodeling factors). Previous experimental studies of collaborative competition analyzed cases in which target sites for pairs of cooperating TFs were contained within the same side of the nucleosome. Here, we utilize new assays to measure cooperativity in protein binding to pairs of nucleosomal DNA target sites. We focus on the cases that are of greatest in vivo relevance, in which one binding site is located close to the end of a nucleosome and the other binding site is located at diverse positions throughout the nucleosome. Our results reveal energetically significant positive (favorable) cooperativity for pairs of sites on the same side of the nucleosome but, for the cases examined, energetically insignificant cooperativity between sites on opposite sides of the nucleosome. These findings imply a special significance for TF binding sites that are spaced within one-half nucleosome length (74 bp) or less along the genome and may prove useful for prediction of cooperatively acting TFs genome wide.
Turner, Bryan M
The nucleosome core particle is the first stage of DNA packaging in virtually all eukaryotes. It both organises nuclear DNA and protects it from adventitious binding of transcription factors and the consequent deregulation of gene expression. Both properties are essential to allow the genome expansion characteristic of complex eukaryotes. The nucleosome is a flexible structure in vivo, allowing selective relaxation of its intrinsically inhibitory effects in response to external signals. Structural changes are brought about by dedicated remodelling enzymes and by posttranslational modifications of the core histones. Histone modifications occasionally alter nucleosome structure directly, but their more usual roles are to act as receptors on the nucleosome surface that are recognised by specific protein domains. The bound proteins, in turn, affect nucleosome structure and function. This strategy enormously expands the signalling capacity of the nucleosome and its ability to influence both the initiation and elongation stages of transcription. The enzymes responsible for placing and removing histone modifications, and the modification-binding proteins themselves, are ubiquitous, numerous and conserved amongst eukaryotes. Like the nucleosome, they date back to the earliest eukaryotes and may have played integral and essential roles in eukaryotic evolution. The present properties and epigenetic functions of the nucleosome reflect its evolutionary past and the selective pressures to which it has responded and can be better understood in this context. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.
Mimoto, Mizuho S; Christian, Jan L
Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect.
Lercher, Lukas; Raj, Ritu; Patel, Nisha A.; Price, Joshua; Mohammed, Shabaz; Robinson, Carol V.; Schofield, Christopher J.; Davis, Benjamin G.
O-GlcNAcylation is a newly discovered histone modification implicated in transcriptional regulation, but no structural information on the physical effect of GlcNAcylation on chromatin exists. Here, we generate synthetic, pure GlcNAcylated histones and nucleosomes and reveal that GlcNAcylation can modulate structure through direct destabilization of H2A/H2B dimers in the nucleosome, thus promoting an `open' chromatin state. The results suggest that a plausible molecular basis for one role of histone O-GlcNAcylation in epigenetic regulation is to lower the barrier for RNA polymerase passage and hence increase transcription.
Lercher, Lukas; Raj, Ritu; Patel, Nisha A.; Price, Joshua; Mohammed, Shabaz; Robinson, Carol V.; Schofield, Christopher J.; Davis, Benjamin G.
O-GlcNAcylation is a newly discovered histone modification implicated in transcriptional regulation, but no structural information on the physical effect of GlcNAcylation on chromatin exists. Here, we generate synthetic, pure GlcNAcylated histones and nucleosomes and reveal that GlcNAcylation can modulate structure through direct destabilization of H2A/H2B dimers in the nucleosome, thus promoting an ‘open' chromatin state. The results suggest that a plausible molecular basis for one role of histone O-GlcNAcylation in epigenetic regulation is to lower the barrier for RNA polymerase passage and hence increase transcription. PMID:26305776
Felle, Max; Hoffmeister, Helen; Rothammer, Julia; Fuchs, Andreas; Exler, Josef H.; Längst, Gernot
Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo. PMID:21622955
Sharma, Vishva Mitra; Li, Bing; Reese, Joseph C.
Gene expression requires the recruitment of chromatin remodeling activities and general transcription factors (GTFs) to promoters. Whereas the role of activators in recruiting chromatin remodeling activities has been clearly demonstrated, the contributions of the transcription machinery have not been firmly established. Here we demonstrate that the remodeling of the RNR3 promoter requires a number of GTFs, mediator and RNA polymerase II. We also show that remodeling is dependent upon the SWI/SNF complex, and that TFIID and RNA polymerase II are required for its recruitment to the promoter. In contrast, Gcn5p-dependent histone acetylation occurs independently of TFIID and RNA polymerase II function, and we provide evidence that acetylation increases the extent of nucleosome remodeling, but is not required for SWI/SNF recruitment. Thus, the general transcription machinery can contribute to nucleosome remodeling by mediating the association of SWI/SNF with promoters, thereby revealing a novel pathway for the recruitment of chromatin remodeling activities. PMID:12600943
Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero
Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ?10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.
Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero
Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell. PMID:25418099
Tomschik, Miroslav; Zheng, Haocheng; van Holde, Ken; Zlatanova, Jordanka; Leuba, Sanford H.
The nucleosome core particle, the basic repeated structure in chromatin fibers, consists of an octamer of eight core histone molecules, organized as dimers (H2A/H2B) and tetramers [(H3/H4)2] around which DNA wraps tightly in almost two left-handed turns. The nucleosome has to undergo certain conformational changes to allow processes that need access to the DNA template to occur. By single-pair fluorescence resonance energy transfer, we demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed), with the DNA wrapped around the histone core, or open, with the DNA significantly unraveled from the histone octamer. The brief excursions into an extended open state may create windows of opportunity for protein factors involved in DNA transactions to bind to or translocate along the DNA. conformational transitions | evanescent field fluorescence microscope | nucleosome dynamics | nucleosome opening
Zalckvar, Einat; Paulus, Christina; Tillo, Desiree; Asbach-Nitzsche, Alexandra; Lubling, Yaniv; Winterling, Carla; Strieder, Nicholas; Mücke, Katrin; Goodrum, Felicia; Segal, Eran; Nevels, Michael
Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function.
Cole, Hope A.; Howard, Bruce H.; Clark, David J.
We have used paired-end sequencing of yeast nucleosomal DNA to obtain accurate genomic maps of nucleosome positions and occupancies in control cells and cells treated with 3-aminotriazole (3AT), an inducer of the transcriptional activator Gcn4. In control cells, 3AT-inducible genes exhibit a series of distinct nucleosome occupancy peaks. However, the underlying position data reveal that each nucleosome peak actually consists of a cluster of mutually exclusive overlapping positions, usually including a dominant position. Thus, each nucleosome occupies one of several possible positions and consequently, different cells have distinct local chromatin structures. Induction results in a major disruption of nucleosome positioning, sometimes with altered spacing and a dramatic loss of occupancy over the entire gene, often extending into a neighbouring gene. Nucleosome-depleted regions are generally unaffected. Genes repressed by 3AT show the same changes, but in reverse. We propose that yeast genes exist in one of several alternative nucleosomal arrays, which are disrupted by activation. We conclude that activation results in gene-wide chromatin remodelling and that this remodelling can even extend into the chromatin of flanking genes. PMID:21880600
Cole, Hope A; Howard, Bruce H; Clark, David J
We have used paired-end sequencing of yeast nucleosomal DNA to obtain accurate genomic maps of nucleosome positions and occupancies in control cells and cells treated with 3-aminotriazole (3AT), an inducer of the transcriptional activator Gcn4. In control cells, 3AT-inducible genes exhibit a series of distinct nucleosome occupancy peaks. However, the underlying position data reveal that each nucleosome peak actually consists of a cluster of mutually exclusive overlapping positions, usually including a dominant position. Thus, each nucleosome occupies one of several possible positions and consequently, different cells have distinct local chromatin structures. Induction results in a major disruption of nucleosome positioning, sometimes with altered spacing and a dramatic loss of occupancy over the entire gene, often extending into a neighbouring gene. Nucleosome-depleted regions are generally unaffected. Genes repressed by 3AT show the same changes, but in reverse. We propose that yeast genes exist in one of several alternative nucleosomal arrays, which are disrupted by activation. We conclude that activation results in gene-wide chromatin remodelling and that this remodelling can even extend into the chromatin of flanking genes. Published by Oxford University Press.
Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F; Kang, Sukhyun; Belsky, Jason A; MacAlpine, David M; Peterson, Craig L; Bell, Stephen P
Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin. DOI: http://dx.doi.org/10.7554/eLife.22512.001 PMID:28322723
McKay, Daniel J; Lieb, Jason D
A high-resolution look at where histones touch DNA reveals a surprisingly intricate, dynamic, and modular nucleosome. Three advances in the study by Rhee et al. include unexpected interactions between the H3 tail and linker DNA, new evidence for existence of subnucleosomal particles, and asymmetric patterns of histone modification within a single nucleosome that correspond to the direction of transcription.
Karau, Kelly L.; Molthen, Robert C.; Johnson, Roger H.; Dhyani, Anita H.; Haworth, Steven T.; Dawson, Christopher A.
Animal models and micro-CT imaging are useful for understanding the functional consequences of, and identifying the genes involved in, the remodeling of vascular structures that accompanies pulmonary vascular disease. Using a micro-CT scanner to image contrast-enhanced arteries in excised lungs from fawn hooded rats (a strain genetically susceptible to hypoxia induced pulmonary hypertension), we found that portions of the pulmonary arterial tree downstream from a given diameter were morphometrically indistinguishable. This 'self-consistency' property provided a means for summarizing the pulmonary arterial tree architecture and mechanical properties using a parameter vector obtained from measurements of the contiguous set of vessel segments comprising the longest (principal) pathway and its branches over a range of vascular pressures. This parameter vector was used to characterize the pulmonary vascular remodeling that occurred in rats exposed to a hypoxic (11.5% oxygen) environment and provided the input to a hemodynamic model relating structure to function. The major effect of the remodeling was a longitudinally (pulmonary artery to arterioles) uniform decrease in vessel distensibility that resulted in a 90% increase in arterial resistance. Despite the almost uniform change in vessel distensibility, over 50% of the resistance increase was attributable to vessels with unstressed diameters less than 125 microns.
Zhang, Yong; Vastenhouw, Nadine L; Feng, Jianxing; Fu, Kai; Wang, Chenfei; Ge, Ying; Pauli, Andrea; van Hummelen, Paul; Schier, Alexander F; Liu, X Shirley
The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of histone H3 lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.
Kavi, Harsh; Lu, Xingwu; Xu, Na; Bartholdy, Boris A.; Vershilova, Elena; Skoultchi, Arthur I.; Fyodorov, Dmitry V.
Chromatin structure and activity can be modified through ATP-dependent repositioning of nucleosomes and posttranslational modifications of core histone tails within nucleosome core particles and by deposition of linker histones into the oligonucleosome fiber. The linker histone H1 is essential in metazoans. It has a profound effect on organization of chromatin into higher-order structures and on recruitment of histone-modifying enzymes to chromatin. Here, we describe a genetic screen for modifiers of the lethal phenotype caused by depletion of H1 in Drosophila melanogaster. We identify 41 mis-expression alleles that enhance and 20 that suppress the effect of His1 depletion in vivo. Most of them are important for chromosome organization, transcriptional regulation, and cell signaling. Specifically, the reduced viability of H1-depleted animals is strongly suppressed by ubiquitous mis-expression of the ATP-dependent chromatin remodeling enzyme CHD1. Comparison of transcript profiles in H1-depleted and Chd1 null mutant larvae revealed that H1 and CHD1 have common transcriptional regulatory programs in vivo. H1 and CHD1 share roles in repression of numerous developmentally regulated and extracellular stimulus-responsive transcripts, including immunity-related and stress response-related genes. Thus, linker histone H1 participates in various regulatory programs in chromatin to alter gene expression. PMID:25628309
Valouev, Anton; Ichikawa, Jeffrey; Tonthat, Thaisan; Stuart, Jeremy; Ranade, Swati; Peckham, Heather; Zeng, Kathy; Malek, Joel A; Costa, Gina; McKernan, Kevin; Sidow, Arend; Fire, Andrew; Johnson, Steven M
Using the massively parallel technique of sequencing by oligonucleotide ligation and detection (SOLiD; Applied Biosystems), we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this data set, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.
Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H
The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome
Salih, Bilal; Tripathi, Vijay; Trifonov, Edward N
Fifteen years ago, Lowary and Widom assembled nucleosomes on synthetic random sequence DNA molecules, selected the strongest nucleosomes and discovered that the TA dinucleotides in these strong nucleosome sequences often appear at 10-11 bases from one another or at distances which are multiples of this period. We repeated this experiment computationally, on large ensembles of natural genomic sequences, by selecting the strongest nucleosomes--i.e. those with such distances between like-named dinucleotides, multiples of 10.4 bases, the structural and sequence period of nucleosome DNA. The analysis confirmed the periodicity of TA dinucleotides in the strong nucleosomes, and revealed as well other periodic sequence elements, notably classical AA and TT dinucleotides. The matrices of DNA bendability and their simple linear forms--nucleosome positioning motifs--are calculated from the strong nucleosome DNA sequences. The motifs are in full accord with nucleosome positioning sequences derived earlier, thus confirming that the new technique, indeed, detects strong nucleosomes. Species- and isochore-specific variations of the matrices and of the positioning motifs are demonstrated. The strong nucleosome DNA sequences manifest the highest hitherto nucleosome positioning sequence signals, showing the dinucleotide periodicities in directly observable rather than in hidden form.
Liu, Ning; Peterson, Craig L; Hayes, Jeffrey J
The multisubunit SWI/SNF and RSC complexes utilize energy derived from ATP hydrolysis to mobilize nucleosomes and render the DNA accessible for various nuclear processes. Here we test the idea that remodeling involves intermediates with mobile DNA bulges or loops within the nucleosome by cross-linking the H2A N- or C-terminal tails together to generate protein "loops" that constrict separation of the DNA from the histone surface. Analyses indicate that this intranucleosomal cross-linking causes little or no change in remodeling-dependent exposure of DNA sequences within the nucleosome to restriction enzymes. However, cross-linking inhibits nucleosome mobilization and blocks complete movement of nucleosomes to extreme end positions on the DNA fragments. These results are consistent with evidence that nucleosome remodeling involves intermediates with DNA loops on the nucleosome surface but indicate that such loops do not freely diffuse about the surface of the histone octamer. We propose a threading model for movement of DNA loops around the perimeter of the nucleosome core.
This dissertation concentrates on the structure of HMG-nucleosome complexes, and how they differ from nucleosomes alone. The first chapter provides an introduction to chromatin and an overview of the field. The second and third chapters describe what kinds of nucleosome-HMG protein complexes form in solution, and where the HMG proteins may bind on the nucleosome. A model is proposed that locates the HMG binding sites on the nucleosome core particle. The fourth chapter describes the biophysical characterization of the complex. The methods include thermal denaturation, circular dichroism and sedimentation velocity, all done under variety of solvent conditions. These methods reveal a great deal of information on the stability and interactions of the complex. The fifth chapter describes conformational probes of the complex. These results reveal the structural transitions that occur when HMG protein binds to the nucleosome as well as the parts of the nucleosome essential for the binding reaction.
Salih, Bilal; Trifonov, Edward N
Earlier identified strongest nucleosome DNA sequences of A. thaliana, those with visible 10-11 base sequence periodicity, are mapped along chromosomes. Resulting positional distributions reveal distinct maxima, one per chromosome, located in the centromere regions. Sequence-directed nucleosome mapping demonstrates that the strong nucleosomes (SNs) make tight arrays, several 'parallel' nucleosomes each, suggesting a columnar chromatin structure. The SNs represent a new class of centromeric nucleosomes, presumably, participating in synapsis of chromatids and securing the centromere architecture.
McKnight, Jeffrey N.; Jenkins, Katherine R.; Nodelman, Ilana M.; Escobar, Thelma; Bowman, Gregory D.
Chd1- and ISWI-type chromatin remodelers can sense extranucleosomal DNA and preferentially shift nucleosomes toward longer stretches of available DNA. The DNA-binding domains of these chromatin remodelers are believed to be responsible for sensing extranucleosomal DNA and are needed for robust sliding, but it is unclear how these domains contribute to directional movement of nucleosomes. Here, we show that the DNA-binding domain of Chd1 is not essential for nucleosome sliding but is critical for centering mononucleosomes on short DNA fragments. Remarkably, nucleosome centering was achieved by replacing the native DNA-binding domain of Chd1 with foreign DNA-binding domains of Escherichia coli AraC or Drosophila melanogaster engrailed. Introducing target DNA sequences recognized by the foreign domains enabled the remodelers to rapidly shift nucleosomes toward these binding sites, demonstrating that these foreign DNA-binding domains dictated the direction of sliding. Sequence-directed sliding occluded the target DNA sequences on the nucleosome enough to promote release of the remodeler. Target DNA sequences were highly stimulatory at multiple positions flanking the nucleosome and had the strongest influence when separated from the nucleosome by 23 or fewer base pairs. These results suggest that the DNA-binding domain's affinity for extranucleosomal DNA is the key determinant for the direction that Chd1 shifts the nucleosome. PMID:21969605
Saha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R
Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications regulate remodeller activity and consider a model for the exposure of nucleosomal DNA that involves the use of directional DNA translocation to pump 'DNA waves' around the nucleosome.
Osberg, Brendan; Nuebler, Johannes; Korber, Philipp; Gerland, Ulrich
The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the 'softness' of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to 'hard' particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.
Usachenko, S.I.; Bradbury, E.M. |
The magnitude of the problem of understanding the structure/function relationships of eukaryotic chromosomes can be appreciated from the fact that the human diploid genome contains more than 2 meters of DNA packaged into 46 chromosomes, each at metaphase being several microns in length. Each chromatid of a chromosome contains a single DNA molecule several centimeters in length. In addition to the DNA, chromosomes contain an equal weight of histones and an equal weight of non-histone chromosomal proteins. These histones are the major chromosomal structural proteins. The non-histone chromosomal proteins are involved in the DNA processes of transcription and replication, in chromosome organization and in nuclear architecture. Polytene chromosomes with their bands and interbands and puffs of active genetic loci provide visual evidence for long range order as do the bands and interbands of mammalian metaphase chromosomes. The gentle removal of histones and all but the most tightly bound 2--3% of non-histone proteins from metaphase chromosomes revealed by electron microscopy a residual protein scaffold constraining a halo of DNA loops extending out from the scaffold.
Berbenetz, Nicolas M.; Nislow, Corey; Brown, Grant W.
Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function. PMID:20824081
The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. A high nucleosome coverage is essential for cells, e.g. to prevent cryptic transcription, and the local positions of specific nucleosomes can play an important role in gene regulation. It is known that in vivo nucleosome positions are affected by a complex mix of passive and active mechanisms, including sequence-specific histone-DNA binding, nucleosome-nucleosome interactions, ATP-dependent remodeling enzymes, transcription, and DNA replication. Yet, the statistical distribution of nucleosome positions is extremely well described by simple physical models that treat the chromatin fiber as an interacting one-dimensional gas. I will discuss how can we interpret this surprising observation from a mechanistic perspective. I will also discuss the kinetics of the interacting gas model, which is pertinent to the question of how cells achieve the high nucleosome coverage within a short time, e.g. after DNA replication.
González, Sara; García, Alicia; Vázquez, Enrique; Serrano, Rebeca; Sánchez, Mar; Quintales, Luis; Antequera, Francisco
In the yeast genome, a large proportion of nucleosomes occupy well-defined and stable positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genome remains controversial. Through quantitative analysis of nucleosome positioning, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes in three species of Schizosaccharomyces and in Saccharomyces cerevisiae. This effect is equally detected in transcribed and nontranscribed regions, suggesting the existence of sequence elements that contribute to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open the possibility of designing coding and noncoding DNA molecules capable of directing their own nucleosomal organization. PMID:27662899
Dalmasso, Maria C.; Onyango, David O.; Naguleswaran, Arunasalam; Sullivan, William J.; Angel, Sergio O.
Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here we report the characterization of three H2A histones, a canonical H2A1 and variants H2AX and H2AZ. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)φ (φ denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We also show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We also found that h2ax expression, but not h2a1 and h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are actively expressed and h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this life cycle stage. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation. PMID:19607843
Tims, Hannah S; Gurunathan, Kaushik; Levitus, Marcia; Widom, Jonathan
Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.
Gkikopoulos, Triantaffyllos; Schofield, Pieta; Singh, Vijender; Pinskaya, Marina; Mellor, Jane; Smolle, Michaela; Workman, Jerry L.; Barton, Geoffrey; Owen-Hughes, Tom
The positioning of nucleosomes within the coding regions of eukaryotic genes is aligned with respect to transcriptional start sites. This organization is likely to influence many genetic processes, requiring access to the underlying DNA. Here we show that the combined action of Isw1 and Chd1 nucleosome spacing enzymes is required to maintain this organization. In the absence of these enzymes regular positioning of the majority of nucleosomes is lost. Exceptions include the region upstream of the promoter, the +1 nucleosome and a subset of locations distributed throughout coding regions where other factors are likely to be involved. These observations indicated that ATP-dependent remodeling enzymes are responsible for directing the positioning of the majority of nucleosomes within the Saccharomyces cerevisiae genome. PMID:21940898
Kukimoto, Iwao; Elderkin, Sarah; Grimaldi, Margaret; Oelgeschläger, Thomas; Varga-Weisz, Patrick D
The histone fold is a structural motif with which two related proteins interact and is found in complexes involved in wrapping DNA, the nucleosome, and transcriptional regulation, as in NC2. We reveal a novel function for histone-fold proteins: facilitation of nucleosome remodeling. ACF1-ISWI complex (ATP-dependent chromatin assembly and remodeling factor [ACF]) associates with histone-fold proteins (CHRAC-15 and CHRAC-17 in the human chromatin accessibility complex [CHRAC]) whose functional relevance has been unclear. We show that these histone-fold proteins facilitate ATP-dependent nucleosome sliding by ACF. Direct interaction of the CHRAC-15/17 complex with the ACF1 subunit is essential for this process. CHRAC-17 interacts with another histone-fold protein, p12, in DNA polymerase epsilon, but CHRAC-15 is essential for interaction with ACF and enhancement of nucleosome sliding. Surprisingly, CHRAC-15/17, p12/CHRAC-17, and NC2 complexes facilitate ACF-mediated chromatin assembly by a mechanism different from nucleosome sliding enhancement, suggesting a general activity of H2A/H2B type histone-fold complexes in chromatin assembly.
Krietenstein, Nils; Wal, Megha; Watanabe, Shinya; Park, Bongsoo; Peterson, Craig L; Pugh, B Franklin; Korber, Philipp
Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization. Copyright © 2016 Elsevier Inc. All rights reserved.
Wippo, Christian J; Krstulovic, Bojana Silic; Ertel, Franziska; Musladin, Sanja; Blaschke, Dorothea; Stürzl, Sabrina; Yuan, Guo-Cheng; Hörz, Wolfram; Korber, Philipp; Barbaric, Slobodan
We showed previously that the strong PHO5 promoter is less dependent on chromatin cofactors than the weaker coregulated PHO8 promoter. In this study we asked if chromatin remodeling at the even stronger PHO84 promoter was correspondingly less cofactor dependent. The repressed PHO84 promoter showed a short hypersensitive region that was flanked upstream and downstream by a positioned nucleosome and contained two transactivator Pho4 sites. Promoter induction generated an extensive hypersensitive and histone-depleted region, yielding two more Pho4 sites accessible. This remodeling was strictly Pho4 dependent, strongly dependent on the remodelers Snf2 and Ino80 and on the histone acetyltransferase Gcn5, and more weakly on the acetyltransferase Rtt109. Importantly, remodeling of each of the two positioned nucleosomes required Snf2 and Ino80 to different degrees. Only remodeling of the upstream nucleosome was strictly dependent on Snf2. Further, remodeling of the upstream nucleosome was more dependent on Ino80 than remodeling of the downstream nucleosome. Both nucleosomes differed in their intrinsic stabilities as predicted in silico and measured in vitro. The causal relationship between the different nucleosome stabilities and the different cofactor requirements was shown by introducing destabilizing mutations in vivo. Therefore, chromatin cofactor requirements were determined by intrinsic nucleosome stabilities rather than correlated to promoter strength.
Nibhani, Reshma; Trifonov, Edward N
Recently developed latest version of the sequence-directed single-base resolution nucleosome mapping reveals existence of strong nucleosomes and chromatin columnar structures (columns). Broad application of this simple technique for further studies of chromatin and chromosome structure requires some basic understanding as to how it works and what information it affords. The paper provides such an introduction to the method. The oscillating maps of singular nucleosomes, of short and long oligonucleosome columns, are explained, as well as maps of chromatin on satellite DNA and occurrences of counter-phase (antiparallel) nucleosome neighbors.
McGhee, J.D.; Felsenfeld, G.; Eisenberg, H.
We have used a variety of chemical probes to measure the accessibility of DNA on the surface of the nucleosome. We review these results, and describe new experiments which show that T4 phage DNA can form complexes with the core histones, possessing the properties of normal nucleosomes. Since T4 DNA is largely occupied by glucose residues in the major groove, this suggests that the major groove is not filled with histone amino acid side chains. We also report results of recent measurements which appear to show that only a few strong charge interactions are involved in the attachment of the terminal 20 nucleotide pairs at each end of nucleosome core DNA. We speculate on the possible functional significance of the accessibility of DNA revealed by all of these experiments. We have also examined conformational changes induced in nucleosomes at high ionic strength (0.5 to 0.7M NaCl). The frictional coefficient is found to undergo a small increase in this region, not consistent with models in which the nucleosome is completely unfolded, but possibly reflecting the dissociation of terminal DNA from the nucleosome surface.
Zhong, Jianling; Luo, Kaixuan; Winter, Peter S; Crawford, Gregory E; Iversen, Edwin S; Hartemink, Alexander J
Although deoxyribonuclease I (DNase I) was used to probe the structure of the nucleosome in the 1960s and 1970s, in the current high-throughput sequencing era, DNase I has mainly been used to study genomic regions devoid of nucleosomes. Here, we reveal for the first time that DNase I can be used to precisely map the (translational) positions of in vivo nucleosomes genome-wide. Specifically, exploiting a distinctive DNase I cleavage profile within nucleosome-associated DNA--including a signature 10.3 base pair oscillation that corresponds to accessibility of the minor groove as DNA winds around the nucleosome--we develop a Bayes-factor-based method that can be used to map nucleosome positions along the genome. Compared to methods that require genetically modified histones, our DNase-based approach is easily applied in any organism, which we demonstrate by producing maps in yeast and human. Compared to micrococcal nuclease (MNase)-based methods that map nucleosomes based on cuts in linker regions, we utilize DNase I cuts both outside and within nucleosomal DNA; the oscillatory nature of the DNase I cleavage profile within nucleosomal DNA enables us to identify translational positioning details not apparent in MNase digestion of linker DNA. Because the oscillatory pattern corresponds to nucleosome rotational positioning, it also reveals the rotational context of transcription factor (TF) binding sites. We show that potential binding sites within nucleosome-associated DNA are often centered preferentially on an exposed major or minor groove. This preferential localization may modulate TF interaction with nucleosome-associated DNA as TFs search for binding sites.
Prasad, Rashmi; D'Arcy, Sheena; Hada, Arjan; Luger, Karolin
The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog in Saccharomyces cerevisiae. Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1. PMID:27273866
Li, Zhaoyu; Gadue, Paul; Chen, Kaifu; Jiao, Yang; Tuteja, Geetu; Schug, Jonathan; Li, Wei; Kaestner, Klaus H.
SUMMARY Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, while DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions. PMID:23260146
Sexton, Brittany S; Druliner, Brooke R; Vera, Daniel L; Avey, Denis; Zhu, Fanxiu; Dennis, Jonathan H
Nucleosome occupancy is critically important in regulating access to the eukaryotic genome. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. We measured nucleosome distributions at high temporal resolution following Kaposi's-sarcoma-associated herpesvirus (KSHV) reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the transcription start sites (TSS) of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. The widespread changes consisted of an indiscriminate remodeling event resulting in the loss of nucleosome rotational phasing signals. Additionally, one in six TSSs in the human genome possessed nucleosomes that are translationally remodeled. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the underlying DNA sequence. Finally we demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes provide a new framework for understanding the role of chromatin in the genomic response, and have allowed us to propose a hierarchical model for chromatin-based regulation of genome response.
Chandy, Mark; Gutiérrez, José L.; Prochasson, Philippe; Workman, Jerry L.
SWI/SNF is a well-characterized chromatin remodeling complex that remodels chromatin by sliding nucleosomes in cis and/or displacing nucleosomes in trans. The latter mechanism has the potential to remove promoter nucleosomes, allowing access to transcription factors and RNA polymerase. In vivo, histone acetylation often precedes apparent nucleosome loss; therefore, we sought to determine whether nucleosomes containing acetylated histones could be displaced by the SWI/SNF chromatin remodeling complex. We found that SAGA-acetylated histones were lost from an immobilized nucleosome array when treated with the SWI/SNF complex. When the nucleosome array was acetylated by SAGA in the presence of bound transcription activators, it generated a peak of acetylation surrounding the activator binding sites. Subsequent SWI/SNF treatment suppressed this acetylation peak. Immunoblots indicated that SWI/SNF preferentially displaced acetylated histones from the array relative to total histones. Moreover, the Swi2/Snf2 bromodomain, an acetyl-lysine binding domain, played a role in the displacement of acetylated histones. These data indicate that targeted histone acetylation by the SAGA complex predisposes promoter nucleosomes for displacement by the SWI/SNF complex. PMID:17030999
da Silva, Isabel Torres Gomes; de Oliveira, Paulo Sergio Lopes; Santos, Guilherme Martins
Chromatin is the major regulator of gene expression and genome maintenance. Proteins that bind the nucleosome, the repetitive unit of chromatin, and the histone H4 tail are critical to establishing chromatin architecture and phenotypic outcomes. Intriguingly, nucleosome-binding proteins (NBPs) and the H4 tail peptide compete for the same binding site at an acidic region on the nucleosome surface. Although the essential facts about the nucleosome were revealed 17 years ago, new insights into its atomic structure and molecular mechanisms are still emerging. Several complex nucleosome:NBP structures were recently revealed, characterizing the NBP-binding sites on the nucleosome surface. Here we discuss the potential of the nucleosome surface as a therapeutic target and the impact and development of exogenous nucleosome-binding molecules (eNBMs).
Voltz, Karine; Trylska, Joanna; Calimet, Nicolas; Smith, Jeremy C.; Langowski, Jörg
To permit access to DNA-binding proteins involved in the control and expression of the genome, the nucleosome undergoes structural remodeling including unwrapping of nucleosomal DNA segments from the nucleosome core. Here we examine the mechanism of DNA dissociation from the nucleosome using microsecond timescale coarse-grained molecular dynamics simulations. The simulations exhibit short-lived, reversible DNA detachments from the nucleosome and long-lived DNA detachments not reversible on the timescale of the simulation. During the short-lived DNA detachments, 9 bp dissociate at one extremity of the nucleosome core and the H3 tail occupies the space freed by the detached DNA. The long-lived DNA detachments are characterized by structural rearrangements of the H3 tail including the formation of a turn-like structure at the base of the tail that sterically impedes the rewrapping of DNA on the nucleosome surface. Removal of the H3 tails causes the long-lived detachments to disappear. The physical consistency of the CG long-lived open state was verified by mapping a CG structure representative of this state back to atomic resolution and performing molecular dynamics as well as by comparing conformation-dependent free energies. Our results suggest that the H3 tail may stabilize the nucleosome in the open state during the initial stages of the nucleosome remodeling process. PMID:22385856
Franklin, Sarah; Chen, Haodong; Mitchell-Jordan, Scherise; Ren, Shuxun; Wang, Yibin; Vondriska, Thomas M
A fundamental question in biology is how genome-wide changes in gene expression are enacted in response to a finite stimulus. Recent studies have mapped changes in nucleosome localization, determined the binding preferences for individual transcription factors, and shown that the genome adopts a nonrandom structure in vivo. What remains unclear is how global changes in the proteins bound to DNA alter chromatin structure and gene expression. We have addressed this question in the mouse heart, a system in which global gene expression and massive phenotypic changes occur without cardiac cell division, making the mechanisms of chromatin remodeling centrally important. To determine factors controlling genomic plasticity, we used mass spectrometry to measure chromatin-associated proteins. We have characterized the abundance of 305 chromatin-associated proteins in normal cells and measured changes in 108 proteins that accompany the progression of heart disease. These studies were conducted on a high mass accuracy instrument and confirmed in multiple biological replicates, facilitating statistical analysis and allowing us to interrogate the data bioinformatically for modules of proteins involved in similar processes. Our studies reveal general principles for global shifts in chromatin accessibility: altered linker to core histone ratio; differing abundance of chromatin structural proteins; and reprogrammed histone post-translational modifications. Using small interfering RNA-mediated loss-of-function in isolated cells, we demonstrate that the non-histone chromatin structural protein HMGB2 (but not HMGB1) suppresses pathologic cell growth in vivo and controls a gene expression program responsible for hypertrophic cell growth. Our findings reveal the basis for alterations in chromatin structure necessary for genome-wide changes in gene expression. These studies have fundamental implications for understanding how global chromatin remodeling occurs with specificity and
Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex.
Kisliouk, Tatiana; Cramer, Tomer; Meiri, Noam
Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter in vitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment in vivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress. © 2017 International Society for Neurochemistry.
Mack, Andrew H; Schlingman, Daniel J; Ilagan, Robielyn P; Regan, Lynne; Mochrie, Simon G J
Chromatin "remodeling" is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA-histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be -32k(B)T for nucleosomes containing unmodified H4 and -27k(B)T for nucleosomes containing H4-R45H. Thus, the "loosening" or "remodeling" caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HO promoter (in the absence of other remodeling factors), is 5k(B)T. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.
Tompitak, M.; de Bruin, L.; Eslami-Mossallam, B.; Schiessel, H.
About three quarters of our DNA is wrapped into nucleosomes: DNA spools with a protein core. It is well known that the affinity of a given DNA stretch to be incorporated into a nucleosome depends on the geometry and elasticity of the basepair sequence involved, causing the positioning of nucleosomes. Here we show that DNA elasticity can have a much deeper effect on nucleosomes than just their positioning: it affects their "identities". Employing a recently developed computational algorithm, the mutation Monte Carlo method, we design nucleosomes with surprising physical characteristics. Unlike any other nucleosomes studied so far, these nucleosomes are short-lived when put under mechanical tension whereas other physical properties are largely unaffected. This suggests that the nucleosome, the most abundant DNA-protein complex in our cells, might more properly be considered a class of complexes with a wide array of physical properties, and raises the possibility that evolution has shaped various nucleosome species according to their genomic context.
Li, Guang; Liu, Shujing; Wang, Jiawei; He, Jianfeng; Huang, Hai; Zhang, Yijing; Xu, Lin
Chromatin is a highly organized structure with repetitive nucleosome subunits. Nucleosome distribution patterns, which contain information on epigenetic controls, are dynamically affected by ATP-dependent chromatin remodeling factors (remodelers). However, whether plants have specific nucleosome distribution patterns and how plant remodelers contribute to the pattern formation are not clear. In this study we used the micrococcal nuclease digestion followed by deep sequencing (MNase-seq) assay to show the genome-wide nucleosome pattern in Arabidopsis thaliana. We demonstrated that the nucleosome distribution patterns of Arabidopsis are associated with the gene expression level, and have several specific characteristics that are different from those of animals and yeast. In addition, we found that remodelers in the A. thaliana imitation switch (AtISWI) subfamily are important for the formation of the nucleosome distribution pattern. Double mutations in the AtISWI genes, CHROMATIN REMODELING 11 (CHR11) and CHR17, resulted in the loss of the evenly spaced nucleosome pattern in gene bodies, but did not affect nucleosome density, supporting a previous idea that the primary role of ISWI is to slide nucleosomes in gene bodies for pattern formation.
Gaykalova, Daria A.; Kulaeva, Olga I.; Volokh, Olesya; Shaytan, Alexey K.; Hsieh, Fu-Kai; Kirpichnikov, Mikhail P.; Sokolova, Olga S.; Studitsky, Vasily M.
Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA–histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone–histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA–protein and protein–protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed. PMID:26460019
Prasad, Punit; Bartholomew, Blaine
A key feature of ATP-dependent chromatin remodeling complexes is how they control the ability of the complex to translocate along DNA within the context of a nucleosome. Although these complexes generally initiate DNA translocation near the dyad axis of the nucleosome, the progression and eventual termination is regulated in quite distinct ways. The best studied examples of these are the ISWI type which has strong extranucleosomal DNA dependent activity or the SWI/SNF type which has no linker DNA requirement. Recent data provide insights into the mechanism of regulation of DNA translocation by the ISWI type complexes and how the structure of the ISWI-nucleosome complex changes during chromatin remodeling.
Chalkley, Gillian E.; Kan, Tsung Wai; Reddy, B. Ashok; Ozgur, Zeliha; van Ijcken, Wilfred F. J.; Dekkers, Dick H. W.; Demmers, Jeroen A.; Travers, Andrew A.
The nucleosome is the fundamental repeating unit of eukaryotic chromatin. Here, we assessed the interplay between DNA sequence and ATP-dependent chromatin-remodeling factors (remodelers) in the nucleosomal organization of a eukaryotic genome. We compared the genome-wide distribution of Drosophila NURD, (P)BAP, INO80, and ISWI, representing the four major remodeler families. Each remodeler has a unique set of genomic targets and generates distinct chromatin signatures. Remodeler loci have characteristic DNA sequence features, predicted to influence nucleosome formation. Strikingly, remodelers counteract DNA sequence-driven nucleosome distribution in two distinct ways. NURD, (P)BAP, and INO80 increase histone density at their target sequences, which intrinsically disfavor positioned nucleosome formation. In contrast, ISWI promotes open chromatin at sites that are propitious for precise nucleosome placement. Remodelers influence nucleosome organization genome-wide, reflecting their high genomic density and the propagation of nucleosome redistribution beyond remodeler binding sites. In transcriptionally silent early embryos, nucleosome organization correlates with intrinsic histone-DNA sequence preferences. Following differential expression of the genome, however, this relationship diminishes and eventually disappears. We conclude that the cellular nucleosome landscape is the result of the balance between DNA sequence-driven nucleosome placement and active nucleosome repositioning by remodelers and the transcription machinery. PMID:22124157
Jimeno-González, Silvia; Ceballos-Chávez, María; Reyes, José C
Chromatin distribution is not uniform along the human genome. In most genes there is a promoter-associated nucleosome free region (NFR) followed by an array of nucleosomes towards the gene body in which the first (+1) nucleosome is strongly positioned. The function of this characteristic chromatin distribution in transcription is not fully understood. Here we show in vivo that the +1 nucleosome plays a role in modulating RNA polymerase II (RNAPII) promoter-proximal pausing. When a +1 nucleosome is strongly positioned, elongating RNAPII has a tendency to stall at the promoter-proximal region, recruits more negative elongation factor (NELF) and produces less mRNA. The nucleosome-induced pause favors pre-mRNA quality control by promoting the addition of the cap to the nascent RNA. Moreover, the uncapped RNAs produced in the absence of a positioned nucleosome are degraded by the 5'-3' exonuclease XRN2. Interestingly, reducing the levels of the chromatin remodeler ISWI factor SNF2H decreases +1 nucleosome positioning and increases RNAPII pause release. This work demonstrates a function for +1 nucleosome in regulation of transcription elongation, pre-mRNA processing and gene expression.
Jimeno-González, Silvia; Ceballos-Chávez, María; Reyes, José C.
Chromatin distribution is not uniform along the human genome. In most genes there is a promoter-associated nucleosome free region (NFR) followed by an array of nucleosomes towards the gene body in which the first (+1) nucleosome is strongly positioned. The function of this characteristic chromatin distribution in transcription is not fully understood. Here we show in vivo that the +1 nucleosome plays a role in modulating RNA polymerase II (RNAPII) promoter-proximal pausing. When a +1 nucleosome is strongly positioned, elongating RNAPII has a tendency to stall at the promoter-proximal region, recruits more negative elongation factor (NELF) and produces less mRNA. The nucleosome-induced pause favors pre-mRNA quality control by promoting the addition of the cap to the nascent RNA. Moreover, the uncapped RNAs produced in the absence of a positioned nucleosome are degraded by the 5′-3′ exonuclease XRN2. Interestingly, reducing the levels of the chromatin remodeler ISWI factor SNF2H decreases +1 nucleosome positioning and increases RNAPII pause release. This work demonstrates a function for +1 nucleosome in regulation of transcription elongation, pre-mRNA processing and gene expression. PMID:25735750
Chen, Weizhong; Liu, Yi; Zhu, Shanshan; Green, Christopher D; Wei, Gang; Han, Jing-Dong Jackie
Accurate determination of genome-wide nucleosome positioning can provide important insights into global gene regulation. Here, we describe the development of an improved nucleosome-positioning algorithm-iNPS-which achieves significantly better performance than the widely used NPS package. By determining nucleosome boundaries more precisely and merging or separating shoulder peaks based on local MNase-seq signals, iNPS can unambiguously detect 60% more nucleosomes. The detected nucleosomes display better nucleosome 'widths' and neighbouring centre-centre distance distributions, giving rise to sharper patterns and better phasing of average nucleosome profiles and higher consistency between independent data subsets. In addition to its unique advantage in classifying nucleosomes by shape to reveal their different biological properties, iNPS also achieves higher significance and lower false positive rates than previously published methods. The application of iNPS to T-cell activation data demonstrates a greater ability to facilitate detection of nucleosome repositioning, uncovering additional biological features underlying the activation process.
Kato, Shigeaki; Fujiki, Ryoji; Kitagawa, Hirochika
We have purified nuclear complexes for Vitamin D receptor (VDR), and identified one of them as a novel ATP-dependent chromatine remodeling containing Williams syndrome transcription factor (WSTF), that is supposed to be responsible for Williams syndrome. This complex (WSTF including nucleosome assembly complex (WINAC)) exhibited an ATP-dependent chromatin remodeling activity in vitro. Transient expression assays revealed that WINAC potentiates ligand-induced function of VDR in gene activation and repression. Thus, this study describes a molecular basis of the VDR function on chromosomal DNA through chromatine remodeling.
Möbius, Wolfram; Gerland, Ulrich
The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in Saccharomyces cerevisiae is qualitatively consistent with a "barrier nucleosome model," in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to approximately 1,000 bp to each side.
Kujirai, Tomoya; Horikoshi, Naoki; Sato, Koichi; Maehara, Kazumitsu; Machida, Shinichi; Osakabe, Akihisa; Kimura, Hiroshi; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi
Histone H3.Y is a primate-specific, distant H3 variant. It is evolutionarily derived from H3.3, and may function in transcription regulation. However, the mechanism by which H3.Y regulates transcription has not been elucidated. In the present study, we determined the crystal structure of the H3.Y nucleosome, and found that many H3.Y-specific residues are located on the entry/exit sites of the nucleosome. Biochemical analyses revealed that the DNA ends of the H3.Y nucleosome were more flexible than those of the H3.3 nucleosome, although the H3.Y nucleosome was stable in vitro and in vivo. Interestingly, the linker histone H1, which compacts nucleosomal DNA, appears to bind to the H3.Y nucleosome less efficiently, as compared to the H3.3 nucleosome. These characteristics of the H3.Y nucleosome are also conserved in the H3.Y/H3.3 heterotypic nucleosome, which may be the predominant form in cells. In human cells, H3.Y preferentially accumulated around transcription start sites (TSSs). Taken together, H3.Y-containing nucleosomes around transcription start sites may form relaxed chromatin that allows transcription factor access, to regulate the transcription status of specific genes. PMID:27016736
Wong, Hua; Victor, Jean-Marc; Mozziconacci, Julien
In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.
Svensson, J Peter; Shukla, Manu; Menendez-Benito, Victoria; Norman-Axelsson, Ulrika; Audergon, Pauline; Sinha, Indranil; Tanny, Jason C; Allshire, Robin C; Ekwall, Karl
Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation. © 2015 Svensson et al.; Published by Cold Spring Harbor Laboratory Press.
Maskell, Daniel P; Renault, Ludovic; Serrao, Erik; Lesbats, Paul; Matadeen, Rishi; Hare, Stephen; Lindemann, Dirk; Engelman, Alan N; Costa, Alessandro; Cherepanov, Peter
Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.
Clauvelin, N; Lo, P; Kulaeva, O I; Nizovtseva, E V; Diaz-Montes, J; Zola, J; Parashar, M; Studitsky, V M; Olson, W K
The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of ∼150 DNA base pairs and eight histone proteins-found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the 'local' inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and
Kono, Hidetoshi; Shirayama, Kazuyoshi; Arimura, Yasuhiro; Tachiwana, Hiroaki; Kurumizaka, Hitoshi
The dynamics of nucleosomes containing either canonical H3 or its centromere-specific variant CENP-A were investigated using molecular dynamics simulations. The simulations showed that the histone cores were structurally stable during simulation periods of 100 ns and 50 ns, while DNA was highly flexible at the entry and exit regions and partially dissociated from the histone core. In particular, approximately 20-25 bp of DNA at the entry and exit regions of the CENP-A nucleosome exhibited larger fluctuations than DNA at the entry and exit regions of the H3 nucleosome. Our detailed analysis clarified that this difference in dynamics was attributable to a difference in two basic amino acids in the αN helix; two arginine (Arg) residues in H3 were substituted by lysine (Lys) residues at the corresponding sites in CENP-A. The difference in the ability to form hydrogen bonds with DNA of these two residues regulated the flexibility of nucleosomal DNA at the entry and exit regions. Our exonuclease III assay consistently revealed that replacement of these two Arg residues in the H3 nucleosome by Lys enhanced endonuclease susceptibility, suggesting that the DNA ends of the CENP-A nucleosome are more flexible than those of the H3 nucleosome. This difference in the dynamics between the two types of nucleosomes may be important for forming higher order structures in different phases.
Chen, Huidong; Guan, Jihong; Zhou, Shuigeng
Nucleosomes and the free linker DNA between them assemble the chromatin. Nucleosome positioning plays an important role in gene transcription regulation, DNA replication and repair, alternative splicing, and so on. With the rapid development of ChIP-seq, it is possible to computationally detect the positions of nucleosomes on chromosomes. However, existing methods cannot provide accurate and detailed information about the detected nucleosomes, especially for the nucleosomes with complex configurations where overlaps and noise exist. Meanwhile, they usually require some prior knowledge of nucleosomes as input, such as the size or the number of the unknown nucleosomes, which may significantly influence the detection results. In this paper, we propose a novel approach DPNuc for identifying nucleosome positions based on the Dirichlet process mixture model. In our method, Markov chain Monte Carlo (MCMC) simulations are employed to determine the mixture model with no need of prior knowledge about nucleosomes. Compared with three existing methods, our approach can provide more detailed information of the detected nucleosomes and can more reasonably reveal the real configurations of the chromosomes; especially, our approach performs better in the complex overlapping situations. By mapping the detected nucleosomes to a synthetic benchmark nucleosome map and two existing benchmark nucleosome maps, it is shown that our approach achieves a better performance in identifying nucleosome positions and gets a higher F-score. Finally, we show that our approach can more reliably detect the size distribution of nucleosomes.
Ye, Youqiong; Gu, Liang; Chen, Xiaolong; Shi, Jiejun; Zhang, Xiaobai; Jiang, Cizhong
Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. Here, we reveal the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells. PMID:27634414
Krajewski, Wladyslaw A
Nucleosome remodeling studies in vitro have primarily focused on the use of mononucleosome templates, which, however, can provide only limited information on how nucleosome mobilization occurs in the context of chromatin, in which internucleosome interactions might influence remodeling. We tried to evaluate whether nucleosome mobilization by yeast Isw1a, Isw1b and Isw2 could be affected by neighboring nucleosomes. We compared mono- and dinucleosomes positioned by the '601' sequence, the studied constructs contain variation in linker length between nucleosomes and variation in the length of flanking sequences. The data characterizing the remodeling were based on gel retardation of the mono and dinucleosomes, keeping in mind the observation that the relative position of the nucleosome will change the mobility of the complex in well defined ways. We found that Isw1a, Isw1b and Isw2 process nucleosomes differently whether they exist as mononucleosomes or dinucleosomes, such as, the Isw1a and Isw1b nucleosome repositioning patterns, which were very similar for mononucleosomes, appeared to be profoundly different in case of dinucleosome templates. We also examined the DNase I protection patterns of remodeled mono- and dinucleosomes. The data suggest that nucleosome mobilizing activity of Isw1a, Isw1b and Isw2 complexes could be significantly influenced by neighboring nucleosomes.
Dindia, Laura A; Alderman, Sarah L; Gillis, Todd E
The changes in the cardiac proteome of rainbow trout (Oncorhynchus mykiss) were quantified during the early phases (4, 7, and 14d) of a typical exercise-training regime to provide a comprehensive overview of the cellular changes responsible for developing a trained heart phenotype. Enhanced somatic growth during the 14d experiment was paralleled by cardiac growth to maintain relative ventricular mass. This was reflected in the cardiac proteome by the increased abundance of contractile proteins and cellular integrity proteins as early as Day 4, including a pronounced and sustained increase in blood vessel epicardial substance - an intercellular adhesion protein expressed in the vertebrate heart. An unexpected finding was that proteins involved in energy pathways, including glycolysis, β-oxidation, the TCA cycle, and the electron transport chain, were generally present at lower levels relative to Day 0 levels, suggesting a reduced investment in the maintenance of energy production pathways. However, as the fish demonstrated somatic and cardiac growth during the exercise-training program, this change did not appear to influence cardiac function. The in-depth analysis of temporal changes in the cardiac proteome of trout during the early stages of exercise training reveals novel insights into cardiac remodelling in an important model species. Rainbow trout hearts have a remarkable ability for molecular, structural, and functional plasticity, and the inherent athleticism of these fish makes them ideal models for studies in comparative exercise physiology. Indeed, several decades of research using exercise-trained trout has shown both conserved and unique aspects of cardiac plasticity induced by a sustained increase in the workload of the heart. Despite a strong appreciation for the outcome of exercise training, however, the temporal events that generate this phenotype are not known. This study interrogates the early stages of exercise training using in-depth proteomic
Krajewski, Wladyslaw A
The three Saccharomyces cerevisiae ISWI chromatin remodeling complexes, Isw1a, Isw1b, and Isw2, are implicated in the regularization of arrayed nucleosomes and regulation of gene activity. Although Isw1a and Isw1b are based on the same catalytic unit, in general, their functions in vivo do not overlap. To better understand the structural consequences of these complexes, we compared the putative nucleosome disrupting activities of the purified Isw1a, Isw1b, and Isw2. To account for the putative effects of nucleosomal environment, we employed reconstituted dinucleosomes in which the histone octamers were specifically positioned by the 146 base pair high-affinity nucleosome sequence "601". We have compared the MNase and deoxyribonuclease I protection patterns of remodeled nucleosome templates and evaluated the nucleosome destabilizing abilities of the Isw1a/b and Isw2 using restriction endonucleases. Although the Isw2 showed little evidence of nucleosome disassembly, the Isw1b remodeled dinucleosomes exhibited some common features with the ySwi-Snf remodeling products. The nuclease digestion data suggest that Isw1a can also promote ATP-dependent distortion of nucleosome structure, although less efficiently than the Isw1b complex.
The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348
The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis.
Lyubchenko, Yuri L.
A fundamental challenge associated with chromosomal gene regulation is accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are dynamic structures rather than static systems, as it was once believed. Direct data is required in order to understand the dynamics of nucleosomes more clearly and answer fundamental questions, including: What is the range of nucleosome dynamics? Does a non-ATP dependent unwrapping process of nucleosomes exist? What are the factors facilitating the large scale opening and unwrapping of nucleosomes? This review summarizes the results of nucleosome dynamics obtained with time-lapse AFM, including a high-speed version (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale. With HS-AFM, the dynamics of nucleosomes at a sub-second time scale was observed allowing one to visualize various pathways of nucleosome dynamics, such as sliding and unwrapping, including complete dissociation. Overall, these findings reveal new insights into the dynamics of nucleosomes and the novel mechanisms controlling spontaneous chromatin dynamics. PMID:24839467
Anné, Jennifer; Edwards, Nicholas P; Wogelius, Roy A; Tumarkin-Deratzian, Allison R; Sellers, William I; van Veelen, Arjen; Bergmann, Uwe; Sokaras, Dimosthenis; Alonso-Mori, Roberto; Ignatyev, Konstantin; Egerton, Victoria M; Manning, Phillip L
Current understanding of bone healing and remodelling strategies in vertebrates has traditionally relied on morphological observations through the histological analysis of thin sections. However, chemical analysis may also be used in such interpretations, as different elements are known to be absorbed and used by bone for different physiological purposes such as growth and healing. These chemical signatures are beyond the detection limit of most laboratory-based analytical techniques (e.g. scanning electron microscopy). However, synchrotron rapid scanning-X-ray fluorescence (SRS-XRF) is an elemental mapping technique that uniquely combines high sensitivity (ppm), excellent sample resolution (20-100 µm) and the ability to scan large specimens (decimetre scale) approximately 3000 times faster than other mapping techniques. Here, we use SRS-XRF combined with microfocus elemental mapping (2-20 µm) to determine the distribution and concentration of trace elements within pathological and normal bone of both extant and extinct archosaurs (Cathartes aura and Allosaurus fragilis). Results reveal discrete chemical inventories within different bone tissue types and preservation modes. Chemical inventories also revealed detail of histological features not observable in thin section, including fine structures within the interface between pathological and normal bone as well as woven texture within pathological tissue.
Anné, Jennifer; Edwards, Nicholas P.; Wogelius, Roy A.; Tumarkin-Deratzian, Allison R.; Sellers, William I.; van Veelen, Arjen; Bergmann, Uwe; Sokaras, Dimosthenis; Alonso-Mori, Roberto; Ignatyev, Konstantin; Egerton, Victoria M.; Manning, Phillip L.
Current understanding of bone healing and remodelling strategies in vertebrates has traditionally relied on morphological observations through the histological analysis of thin sections. However, chemical analysis may also be used in such interpretations, as different elements are known to be absorbed and used by bone for different physiological purposes such as growth and healing. These chemical signatures are beyond the detection limit of most laboratory-based analytical techniques (e.g. scanning electron microscopy). However, synchrotron rapid scanning–X-ray fluorescence (SRS–XRF) is an elemental mapping technique that uniquely combines high sensitivity (ppm), excellent sample resolution (20–100 µm) and the ability to scan large specimens (decimetre scale) approximately 3000 times faster than other mapping techniques. Here, we use SRS–XRF combined with microfocus elemental mapping (2–20 µm) to determine the distribution and concentration of trace elements within pathological and normal bone of both extant and extinct archosaurs (Cathartes aura and Allosaurus fragilis). Results reveal discrete chemical inventories within different bone tissue types and preservation modes. Chemical inventories also revealed detail of histological features not observable in thin section, including fine structures within the interface between pathological and normal bone as well as woven texture within pathological tissue. PMID:24806709
Fennessy, Ross T.; Owen-Hughes, Tom
Nucleosomes, the fundamental subunits of eukaryotic chromatin, are organized with respect to transcriptional start sites. A major challenge to the persistence of this organization is the disassembly of nucleosomes during DNA replication. Here, we use complimentary approaches to map the locations of nucleosomes on recently replicated DNA. We find that nucleosomes are substantially realigned with promoters during the minutes following DNA replication. As a result, the nucleosomal landscape is largely re-established before newly replicated chromosomes are partitioned into daughter cells and can serve as a platform for the re-establishment of gene expression programmes. When the supply of histones is disrupted through mutation of the chaperone Caf1, a promoter-based architecture is generated, but with increased inter-nucleosomal spacing. This indicates that the chromatin remodelling enzymes responsible for spacing nucleosomes are capable of organizing nucleosomes with a range of different linker DNA lengths. PMID:27106059
Petty, Emily; Pillus, Lorraine
Chromatin remodelers use the energy of ATP hydrolysis to reposition or evict nucleosomes or to replace canonical histones with histone variants. By regulating nucleosome dynamics, remodelers gate access to the underlying DNA for replication, repair, and transcription. Nucleosomes are subject to extensive post-translational modifications that can recruit regulatory proteins or alter the local chromatin structure. Just as extensive cross-talk has been observed between different histone post-translational modifications, there is growing evidence for both coordinated and antagonistic functional relationships between nucleosome remodeling and modifying machineries. Defining the combined functions of the complexes that alter nucleosome interactions, position, and stability is key to understanding processes that require access to DNA, particularly with growing appreciation of their contributions to human health and disease. Here, we highlight recent advances in the interactions between histone modifications and the ISWI and CHD1 chromatin remodelers from studies in budding yeast, fission yeast, flies, and mammalian cells, with a focus on yeast. PMID:23870137
Clauvelin, N.; Lo, P.; Kulaeva, O. I.; Nizovtseva, E. V.; Diaz-Montes, J.; Zola, J.; Parashar, M.; Studitsky, V. M.; Olson, W. K.
The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes—the familiar assemblies of ˜150 DNA base pairs and eight histone proteins—found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the ‘local’ inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome
Eukaryotic genomes are organized into arrays of nucleosomes, in which stretches of 147 base-pairs of DNA are wrapped around octameric histones. Recently, a new method of mapping nucleosome positions was developed, which gives a much higher accuracy than the typical MNase-seq method. I present a statistical mechanics model which is able to reproduce the high-resolution nucleosome positioning data. I show that the DNA sequence is not the main cause of the nucleosome phasing which is observed genome-wide, and I present the major nucleosome phasing elements. The statistical mechanics framework is general enough to be useful in explaining different experimental observations, and I present a few results of this model.
Pedone, Francesco; Santoni, Daniele
Nucleosomes are the basic structural units of eukaryotic chromatin and play a key role in the regulation of gene expression. Nucleosome formation depends on several factors, including properties of the sequence itself, but also physical constraints and epigenetic factors such as chromatin-remodelling enzymes. In this view, a sequence-dependent approach is able to capture a general tendency of a region to bind a histone octamer. A reference data set of positioned nucleosomes of Saccharomyces cerevisiae was used to study the role of DNA helical rise in histone–DNA interaction. Genomic sequences were transformed into arrays of helical rise values by a tetranucleotide code and then turned into profiles of mean helical rise values. These profiles resemble maps of nucleosome occupancy, suggesting that intrinsic histone–DNA interactions are linked to helical rise. The obtained results show that preferential nucleosome occupancy occurs where the mean helical rise reaches its largest values. Mean helical rise profiles obtained by using maps of positioned nucleosomes of the Drosophila melanogaster and Plasmodium falciparum genomes, as well as Homo sapiens chromosome 20 confirm that nucleosomes are mainly located where the mean helical rise reaches its largest values. PMID:22233711
Selth, Luke A; Lorch, Yahli; Ocampo-Hafalla, Maria T; Mitter, Richard; Shales, Michael; Krogan, Nevan J; Kornberg, Roger D; Svejstrup, Jesper Q
The histone chaperone Vps75 forms a complex with, and stimulates the activity of, the histone acetyltransferase Rtt109. However, Vps75 can also be isolated on its own and might therefore possess Rtt109-independent functions. Analysis of epistatic miniarray profiles showed that VPS75 genetically interacts with factors involved in transcription regulation whereas RTT109 clusters with genes linked to DNA replication/repair. Additional genetic and biochemical experiments revealed a close relationship between Vps75 and RNA polymerase II. Furthermore, Vps75 is recruited to activated genes in an Rtt109-independent manner, and its genome-wide association with genes correlates with transcription rate. Expression microarray analysis identified a number of genes whose normal expression depends on VPS75. Interestingly, histone H2B dynamics at some of these genes are consistent with a role for Vps75 in histone H2A/H2B eviction/deposition during transcription. Indeed, reconstitution of nucleosome disassembly using the ATP-dependent chromatin remodeler Rsc and Vps75 revealed that these proteins can cooperate to remove H2A/H2B dimers from nucleosomes. These results indicate a role for Vps75 in nucleosome dynamics during transcription, and importantly, this function appears to be largely independent of Rtt109.
Chereji, Razvan V.
Eukaryotic cells contain long DNA molecules (about two meters for a human cell) which are tightly packed inside the micrometric nuclei. Nucleosomes are the basic packaging unit of the DNA which allows this millionfold compactification. A longstanding puzzle is to understand the principles which allow cells to both organize their genomes into chromatin fibers in the crowded space of their nuclei, and also to keep the DNA accessible to many factors and enzymes. With the nucleosomes covering about three quarters of the DNA, their positions are essential because these influence which genes can be regulated by the transcription factors and which cannot. We study physical models which predict the genome-wide organization of the nucleosomes and also the relevant energies which dictate this organization. In the last five years, the study of chromatin knew many important advances. In particular, in the field of nucleosome positioning, new techniques of identifying nucleosomes and the competing DNA-binding factors appeared, as chemical mapping with hydroxyl radicals, ChIP-exo, among others, the resolution of the nucleosome maps increased by using paired-end sequencing, and the price of sequencing an entire genome decreased. We present a rigorous statistical mechanics model which is able to explain the recent experimental results by taking into account nucleosome unwrapping, competition between different DNA-binding proteins, and both the interaction between histones and DNA, and between neighboring histones. We show a series of predictions of our new model, all in agreement with the experimental observations.
Liu, Ning; Balliano, Angela; Hayes, Jeffrey J
Impediments to DNA access due to assembly of the eukaryotic genome into chromatin are in part overcome by the activity of ATP-dependent chromatin-remodeling complexes. These complexes employ energy derived from ATP hydrolysis to destabilize histone-DNA interactions and alter nucleosome positions, thereby increasing the accessibility of DNA-binding factors to their targets. However, the mechanism by which theses complexes accomplish this task remains unresolved. We review aspects of nucleosome alteration by the SWI/SNF complex, the archetypal remodeling enzyme. We focus on experiments that provide insights into how SWI/SNF induces nucleosome movement along DNA. Numerous biochemical activities have been characterized for this complex, all likely providing clues as to the molecular mechanism of translocation.
Schwab, David; Bruinsma, Robijn
The role of chromatin structure in gene regulation has recently taken center stage in the field of epigenetics, phenomena that change the phenotype without changing the DNA sequence. Recent work has also shown that nucleosomes, a complex of DNA wrapped around a histone octamer, experience a sequence dependent energy landscape due to the variation in DNA bend stiffness with sequence composition. In this talk, we consider the role nucleosome positioning might play in the formation of heterochromatin, a compact form of DNA generically responsible for gene silencing. In particular, we discuss how different patterns of nucleosome positions, periodic or random, could either facilitate or suppress heterochromatin stability and formation.
de Boer, Carl G; Hughes, Timothy R
Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.
Baumann, Matthias; Mamais, Adamantios; McBlane, Fraser; Xiao, Hua; Boyes, Joan
A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination. PMID:14517257
Baumann, Matthias; Mamais, Adamantios; McBlane, Fraser; Xiao, Hua; Boyes, Joan
A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.
Ricci, Maria Aurelia; Manzo, Carlo; García-Parajo, María Filomena; Lakadamyali, Melike; Cosma, Maria Pia
Nucleosomes help structure chromosomes by compacting DNA into fibers. To gain insight into how nucleosomes are arranged in vivo, we combined quantitative super-resolution nanoscopy with computer simulations to visualize and count nucleosomes along the chromatin fiber in single nuclei. Nucleosomes assembled in heterogeneous groups of varying sizes, here termed "clutches," and these were interspersed with nucleosome-depleted regions. The median number of nucleosomes inside clutches and their compaction defined as nucleosome density were cell-type-specific. Ground-state pluripotent stem cells had, on average, less dense clutches containing fewer nucleosomes and clutch size strongly correlated with the pluripotency potential of induced pluripotent stem cells. RNA polymerase II preferentially associated with the smallest clutches while linker histone H1 and heterochromatin were enriched in the largest ones. Our results reveal how the chromatin fiber is formed at nanoscale level and link chromatin fiber architecture to stem cell state.
In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While nucleosome positions in vitro are determined by sequence alone, in vivo competition with other DNA-binding factors and action of chromatin remodeling enzymes play a role that needs to be quantified. We developed a biophysical, DNA mechanics-based model for the sequence dependence of DNA bending energies, and validated it against a collection of in vitro free energies of nucleosome formation and a nucleosome crystal structure; we also successfully designed both strong and poor histone binding sequences ab initio. For in vivo data from S.cerevisiae, the strongest positioning signal came from the competition with other factors rather than intrinsic nucleosome sequence preferences. Based on sequence alone, our model predicts that functional transcription factor binding sites tend to be covered by nucleosomes, yet are uncovered in vivo because functional sites cluster within a single nucleosome footprint and thus make transcription factors bind cooperatively. Similarly a weak enhancement of nucleosome binding in the TATA region becomes a strong depletion when the TATA-binding protein is included, in quantitative agreement with experiment. Our model distinguishes multiple ways in which genomic sequence influences nucleosome positions, and thus provides alternative explanations for several genome-wide experimental findings. In the future our approach will be used to rationally alter gene expression levels in model systems through redesign of nucleosome occupancy profiles.
Beshnova, Daria A.; Cherstvy, Andrey G.; Vainshtein, Yevhen; Teif, Vladimir B.
The nucleosome repeat length (NRL) is an integral chromatin property important for its biological functions. Recent experiments revealed several conflicting trends of the NRL dependence on the concentrations of histones and other architectural chromatin proteins, both in vitro and in vivo, but a systematic theoretical description of NRL as a function of DNA sequence and epigenetic determinants is currently lacking. To address this problem, we have performed an integrative biophysical and bioinformatics analysis in species ranging from yeast to frog to mouse where NRL was studied as a function of various parameters. We show that in simple eukaryotes such as yeast, a lower limit for the NRL value exists, determined by internucleosome interactions and remodeler action. For higher eukaryotes, also the upper limit exists since NRL is an increasing but saturating function of the linker histone concentration. Counterintuitively, smaller H1 variants or non-histone architectural proteins can initiate larger effects on the NRL due to entropic reasons. Furthermore, we demonstrate that different regimes of the NRL dependence on histone concentrations exist depending on whether DNA sequence-specific effects dominate over boundary effects or vice versa. We consider several classes of genomic regions with apparently different regimes of the NRL variation. As one extreme, our analysis reveals that the period of oscillations of the nucleosome density around bound RNA polymerase coincides with the period of oscillations of positioning sites of the corresponding DNA sequence. At another extreme, we show that although mouse major satellite repeats intrinsically encode well-defined nucleosome preferences, they have no unique nucleosome arrangement and can undergo a switch between two distinct types of nucleosome positioning. PMID:24992723
Beshnova, Daria A; Cherstvy, Andrey G; Vainshtein, Yevhen; Teif, Vladimir B
The nucleosome repeat length (NRL) is an integral chromatin property important for its biological functions. Recent experiments revealed several conflicting trends of the NRL dependence on the concentrations of histones and other architectural chromatin proteins, both in vitro and in vivo, but a systematic theoretical description of NRL as a function of DNA sequence and epigenetic determinants is currently lacking. To address this problem, we have performed an integrative biophysical and bioinformatics analysis in species ranging from yeast to frog to mouse where NRL was studied as a function of various parameters. We show that in simple eukaryotes such as yeast, a lower limit for the NRL value exists, determined by internucleosome interactions and remodeler action. For higher eukaryotes, also the upper limit exists since NRL is an increasing but saturating function of the linker histone concentration. Counterintuitively, smaller H1 variants or non-histone architectural proteins can initiate larger effects on the NRL due to entropic reasons. Furthermore, we demonstrate that different regimes of the NRL dependence on histone concentrations exist depending on whether DNA sequence-specific effects dominate over boundary effects or vice versa. We consider several classes of genomic regions with apparently different regimes of the NRL variation. As one extreme, our analysis reveals that the period of oscillations of the nucleosome density around bound RNA polymerase coincides with the period of oscillations of positioning sites of the corresponding DNA sequence. At another extreme, we show that although mouse major satellite repeats intrinsically encode well-defined nucleosome preferences, they have no unique nucleosome arrangement and can undergo a switch between two distinct types of nucleosome positioning.
Oka, Toru; Xu, Jian; Kaiser, Robert A.; Melendez, Jaime; Hambleton, Michael; Sargent, Michelle A.; Lorts, Angela; Brunskill, Eric W.; Dorn, Gerald W.; Conway, Simon J.; Aronow, Bruce J.; Robbins, Jeffrey; Molkentin, Jeffery D.
The cardiac extracellular matrix is a dynamic structural support network that is both influenced by, and a regulator of, pathological remodeling and hypertrophic growth. In response to pathologic insults the adult heart re-expresses the secreted extracellular matrix protein periostin (Pn). Here we show that Pn is critically involved in regulating the cardiac hypertrophic response, interstitial fibrosis, and ventricular remodeling following long-term pressure overload stimulation and myocardial infarction. Mice lacking the gene encoding Pn (Postn) were more prone to ventricular rupture in the first 10 days after a myocardial infarction, but surviving mice showed less fibrosis and better ventricular performance. Pn−/− mice also showed less fibrosis and hypertrophy following long-term pressure overload, suggesting an intimate relationship between Pn and the regulation of cardiac remodeling. In contrast, inducible overexpression of Pn in the heart protected mice from rupture following myocardial infarction and induced spontaneous hypertrophy with aging. With respect to a mechanism underlying these alterations, Pn−/− hearts showed an altered molecular program in fibroblast function. Indeed, fibroblasts isolated from Pn−/− hearts were less effective in adherence to cardiac myocytes and were characterized by a dramatic alteration in global gene expression (7% of all genes). These are the first genetic data detailing the function of Pn in the adult heart as a regulator of cardiac remodeling and hypertrophy. PMID:17569887
Williams, Steven P; Odell, Adam F; Karnezis, Tara; Farnsworth, Rae H; Gould, Cathryn M; Li, Jason; Paquet-Fifield, Sophie; Harris, Nicole C; Walter, Anne; Gregory, Julia L; Lamont, Sara F; Liu, Ruofei; Takano, Elena A; Nowell, Cameron J; Bower, Neil I; Resnick, Daniel; Smyth, Gordon K; Coultas, Leigh; Hogan, Benjamin M; Fox, Stephen B; Mueller, Scott N; Simpson, Kaylene J; Achen, Marc G; Stacker, Steven A
Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration. We compared our data set with mRNA differential expression data from endothelial and stromal cells derived from two in vivo models of lymphatic vessel remodeling, viral infection and contact hypersensitivity-induced inflammation, which identified genes selectively involved in regulating LEC migration and remodeling. We also characterized the top candidates in the LEC migratome in primary blood vascular endothelial cells to identify genes with functions common to lymphatic and blood vascular endothelium. On the basis of these analyses, we showed that LGALS1, which encodes the glycan-binding protein Galectin-1, promoted lymphatic vascular growth in vitro and in vivo and contributed to maintenance of the lymphatic endothelial phenotype. Our results provide insight into the signaling networks that control lymphangiogenesis and lymphatic remodeling and potentially identify therapeutic targets and biomarkers in disease specific to lymphatic or blood vessels. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Ozonov, Evgeniy A.; van Nimwegen, Erik
Because DNA packaging in nucleosomes modulates its accessibility to transcription factors (TFs), unraveling the causal determinants of nucleosome positioning is of great importance to understanding gene regulation. Although there is evidence that intrinsic sequence specificity contributes to nucleosome positioning, the extent to which other factors contribute to nucleosome positioning is currently highly debated. Here we obtained both in vivo and in vitro reference maps of positions that are either consistently covered or free of nucleosomes across multiple experimental data-sets in Saccharomyces cerevisiae. We then systematically quantified the contribution of TF binding to nucleosome positiong using a rigorous statistical mechanics model in which TFs compete with nucleosomes for binding DNA. Our results reconcile previous seemingly conflicting results on the determinants of nucleosome positioning and provide a quantitative explanation for the difference between in vivo and in vitro positioning. On a genome-wide scale, nucleosome positioning is dominated by the phasing of nucleosome arrays over gene bodies, and their positioning is mainly determined by the intrinsic sequence preferences of nucleosomes. In contrast, larger nucleosome free regions in promoters, which likely have a much more significant impact on gene expression, are determined mainly by TF binding. Interestingly, of the 158 yeast TFs included in our modeling, we find that only 10–20 significantly contribute to inducing nucleosome-free regions, and these TFs are highly enriched for having direct interations with chromatin remodelers. Together our results imply that nucleosome free regions in yeast promoters results from the binding of a specific class of TFs that recruit chromatin remodelers. PMID:23990766
Ozonov, Evgeniy A; van Nimwegen, Erik
Because DNA packaging in nucleosomes modulates its accessibility to transcription factors (TFs), unraveling the causal determinants of nucleosome positioning is of great importance to understanding gene regulation. Although there is evidence that intrinsic sequence specificity contributes to nucleosome positioning, the extent to which other factors contribute to nucleosome positioning is currently highly debated. Here we obtained both in vivo and in vitro reference maps of positions that are either consistently covered or free of nucleosomes across multiple experimental data-sets in Saccharomyces cerevisiae. We then systematically quantified the contribution of TF binding to nucleosome positioning using a rigorous statistical mechanics model in which TFs compete with nucleosomes for binding DNA. Our results reconcile previous seemingly conflicting results on the determinants of nucleosome positioning and provide a quantitative explanation for the difference between in vivo and in vitro positioning. On a genome-wide scale, nucleosome positioning is dominated by the phasing of nucleosome arrays over gene bodies, and their positioning is mainly determined by the intrinsic sequence preferences of nucleosomes. In contrast, larger nucleosome free regions in promoters, which likely have a much more significant impact on gene expression, are determined mainly by TF binding. Interestingly, of the 158 yeast TFs included in our modeling, we find that only 10-20 significantly contribute to inducing nucleosome-free regions, and these TFs are highly enriched for having direct interactions with chromatin remodelers. Together our results imply that nucleosome free regions in yeast promoters results from the binding of a specific class of TFs that recruit chromatin remodelers.
Silberhorn, Elisabeth; Schwartz, Uwe; Symelka, Anne; de Koning-Ward, Tania; Längst, Gernot
The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome. PMID:28033404
Silberhorn, Elisabeth; Schwartz, Uwe; Löffler, Patrick; Schmitz, Samuel; Symelka, Anne; de Koning-Ward, Tania; Merkl, Rainer; Längst, Gernot
The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome.
Hartley, Paul D.; Madhani, Hiten D.
SUMMARY The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. Here we show that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, we show that NFR establishment is necessary for H2A.Z deposition. These studies suggest an ordered pathway for the assembly of promoter chromatin architecture. PMID:19410542
Kharerin, Hungyo; Bhat, Paike J; Marko, John F; Padinhateeri, Ranjith
Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non-histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different "promoter states" having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions-Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA.
Kharerin, Hungyo; Bhat, Paike J.; Marko, John F.; Padinhateeri, Ranjith
Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non–histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different “promoter states” having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions—Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA. PMID:26843321
Kharerin, Hungyo; Bhat, Paike J.; Marko, John F.; Padinhateeri, Ranjith
Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non-histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different “promoter states” having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions—Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA.
Gutiérrez, José L; Chandy, Mark; Carrozza, Michael J; Workman, Jerry L
ATP-dependent chromatin remodeling complexes play a critical role in chromatin dynamics. A large number of in vitro studies have pointed towards nucleosome sliding as the principal remodeling outcome of SWI/SNF action, whereas few have described histone octamer transfer as the principal outcome. In contrast, recent in vivo studies have linked the activity of SWI/SNF to histone eviction in trans from gene promoters. In this study, we have found that the chimeric transcription factor Gal4-VP16 can enhance SWI/SNF histone octamer transfer activity, resulting in targeted histone eviction from a nucleosome probe. This effect is dependent on the presence of the activation domain. We observed that under conditions mimicking the in vivo relative abundance of SWI/SNF with respect to the total number of nucleosomes in a cell nucleus, the accessibility of the transcription factor binding site is the first determinant in the sequence of events leading to nucleosome remodeling. We propose a model mechanism for this transcription factor-mediated enhancement of SWI/SNF octamer transfer activity. PMID:17235287
Poirier, Michael G; Oh, Eugene; Tims, Hannah S; Widom, Jonathan
The packaging of eukaryotic DNA into chromatin sterically occludes polymerases, recombinases and repair enzymes. How chromatin structure changes to allow their actions is unknown. We constructed defined fluorescently labeled trinucleosome arrays, allowing analysis of chromatin conformational dynamics via fluorescence resonance energy transfer (FRET). The arrays undergo reversible Mg2+-dependent folding similar to that of longer arrays studied previously. We define two intermediate conformational states in the reversible folding of the nucleosome arrays and characterize the microscopic rate constants. Nucleosome arrays are highly dynamic even when compact, undergoing conformational fluctuations on timescales in the second to microsecond range. Compact states of the arrays allow binding to DNA within the central nucleosome via site exposure. Protein binding can also drive decompaction of the arrays. Thus, our results reveal multiple modes by which spontaneous chromatin fiber dynamics allow for the invasion and action of DNA-processing protein complexes.
Ichikawa, Yuichi; Connelly, Caitlin F; Appleboim, Alon; Miller, Thomas Cr; Jacobi, Hadas; Abshiru, Nebiyu A; Chou, Hsin-Jung; Chen, Yuanyuan; Sharma, Upasna; Zheng, Yupeng; Thomas, Paul M; Chen, Hsuiyi V; Bajaj, Vineeta; Müller, Christoph W; Kelleher, Neil L; Friedman, Nir; Bolon, Daniel Na; Rando, Oliver J; Kaufman, Paul D
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read , we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read by effector proteins in the cell.
Onufriev, Alexey; Fenley, Andrew; Zmuda-Ruscio, Jory; Adams, David
The primary level of DNA compaction in eukaryotic organisms is the nucleosome, yet details of its dynamics are not fully understood. While the whole nucleosome must be highly stable, protective of its genetic material, at the same time its tightly wrapped DNA should be highly accessible, easily revealing its information content. A combination of atom-level classical molecular dynamics and a course-grained continuum description provide insights into the functioning of the system. In particular, the nucleosomal DNA appears to be considerably more flexible than what can be expected based on its canonical persistence length. A coarse-grained electrostatic model of the nucleosome explains how its stability can be modulated with small environmental changes as well as post-translational modifications. Implications for the nucleosome assembly process in vivo are discussed.
Chereji, Răzvan V; Kan, Tsung-Wai; Grudniewska, Magda K; Romashchenko, Alexander V; Berezikov, Eugene; Zhimulev, Igor F; Guryev, Victor; Morozov, Alexandre V; Moshkin, Yuri M
Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to 'phasing' off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.
Lequieu, Joshua; Cordoba, Andres; Schwartz, David C.; ...
Here, nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping. Using detailed simulations of DNA and histone proteins, we calculate the tension-dependent free energy surface corresponding to the unwrapping process. The model reproduces quantitatively the forces required to unwrapmore » the nucleosome and reveals the role played by electrostatic interactions during this process. We then demonstrate that histone modifications and DNA sequence can have significant effects on the energies of nucleosome formation. Most notably, we show that histone tails contribute asymmetrically to the stability of the outer and inner turn of nucleosomal DNA and that depending on which histone tails are modified, the tension-dependent response is modulated differently.« less
Lequieu, Joshua; Cordoba, Andres; Schwartz, David C.; de Pablo, Juan J.
Here, nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping. Using detailed simulations of DNA and histone proteins, we calculate the tension-dependent free energy surface corresponding to the unwrapping process. The model reproduces quantitatively the forces required to unwrap the nucleosome and reveals the role played by electrostatic interactions during this process. We then demonstrate that histone modifications and DNA sequence can have significant effects on the energies of nucleosome formation. Most notably, we show that histone tails contribute asymmetrically to the stability of the outer and inner turn of nucleosomal DNA and that depending on which histone tails are modified, the tension-dependent response is modulated differently.
Funke, Jonas J; Ketterer, Philip; Lieleg, Corinna; Schunter, Sarah; Korber, Philipp; Dietz, Hendrik
Revealing the energy landscape for nucleosome association may contribute to the understanding of higher-order chromatin structures and their impact on genome regulation. We accomplish this in a direct measurement by integrating two nucleosomes into a DNA origami-based force spectrometer, which enabled subnanometer-resolution measurements of nucleosome-nucleosome distance frequencies via single-particle electron microscopy imaging. From the data, we derived the Boltzmann-weighted distance-dependent energy landscape for nucleosome pair interactions. We find a shallow but long-range (~6 nm) attractive nucleosome pair potential with a minimum of -1.6 kcal/mol close to direct contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Because of the weak and shallow pair potential, higher-order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional cofactors. Our results contribute to a more accurate description of chromatin and our force spectrometer provides a powerful tool for the direct and high-resolution study of molecular interactions using imaging techniques.
Funke, Jonas J.; Ketterer, Philip; Lieleg, Corinna; Schunter, Sarah; Korber, Philipp; Dietz, Hendrik
Revealing the energy landscape for nucleosome association may contribute to the understanding of higher-order chromatin structures and their impact on genome regulation. We accomplish this in a direct measurement by integrating two nucleosomes into a DNA origami–based force spectrometer, which enabled subnanometer-resolution measurements of nucleosome-nucleosome distance frequencies via single-particle electron microscopy imaging. From the data, we derived the Boltzmann-weighted distance-dependent energy landscape for nucleosome pair interactions. We find a shallow but long-range (~6 nm) attractive nucleosome pair potential with a minimum of −1.6 kcal/mol close to direct contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Because of the weak and shallow pair potential, higher-order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional cofactors. Our results contribute to a more accurate description of chromatin and our force spectrometer provides a powerful tool for the direct and high-resolution study of molecular interactions using imaging techniques. PMID:28138524
Nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping. Using detailed simulations of DNA and histone proteins, we calculate the tension-dependent free energy surface corresponding to the unwrapping process. The model reproduces quantitatively the forces required to unwrap the nucleosome and reveals the role played by electrostatic interactions during this process. We then demonstrate that histone modifications and DNA sequence can have significant effects on the energies of nucleosome formation. Most notably, we show that histone tails contribute asymmetrically to the stability of the outer and inner turn of nucleosomal DNA and that depending on which histone tails are modified, the tension-dependent response is modulated differently. PMID:27725965
Lee, Byoungkoo; Konen, Jessica; Wilkinson, Scott; Marcus, Adam I; Jiang, Yi
Invasive cancer cells interact with the surrounding extracellular matrix (ECM), remodeling ECM fiber network structure by condensing, degrading, and aligning these fibers. We developed a novel local alignment vector analysis method to quantitatively measure collagen fiber alignment as a vector field using Circular Statistics. This method was applied to human non-small cell lung carcinoma (NSCLC) cell lines, embedded as spheroids in a collagen gel. Collagen remodeling was monitored using second harmonic generation imaging under normal conditions and when the LKB1-MARK1 pathway was disrupted through RNAi-based approaches. The results showed that inhibiting LKB1 or MARK1 in NSCLC increases the collagen fiber alignment and captures outward alignment vectors from the tumor spheroid, corresponding to high invasiveness of LKB1 mutant cancer cells. With time-lapse imaging of ECM micro-fiber morphology, the local alignment vector can measure the dynamic signature of invasive cancer cell activity and cell-migration-induced ECM and collagen remodeling and realigning dynamics.
Lee, Byoungkoo; Konen, Jessica; Wilkinson, Scott; Marcus, Adam I.; Jiang, Yi
Invasive cancer cells interact with the surrounding extracellular matrix (ECM), remodeling ECM fiber network structure by condensing, degrading, and aligning these fibers. We developed a novel local alignment vector analysis method to quantitatively measure collagen fiber alignment as a vector field using Circular Statistics. This method was applied to human non-small cell lung carcinoma (NSCLC) cell lines, embedded as spheroids in a collagen gel. Collagen remodeling was monitored using second harmonic generation imaging under normal conditions and when the LKB1-MARK1 pathway was disrupted through RNAi-based approaches. The results showed that inhibiting LKB1 or MARK1 in NSCLC increases the collagen fiber alignment and captures outward alignment vectors from the tumor spheroid, corresponding to high invasiveness of LKB1 mutant cancer cells. With time-lapse imaging of ECM micro-fiber morphology, the local alignment vector can measure the dynamic signature of invasive cancer cell activity and cell-migration-induced ECM and collagen remodeling and realigning dynamics. PMID:28045069
LeGresley, Sarah E.; Wilt, Jamie; Antonik, Matthew
One issue in genome maintenance is how DNA repair proteins find lesions at rates that seem to exceed diffusion-limited search rates. We propose a phenomenon where DNA damage induces nucleosomal rearrangements which move lesions to potential rendezvous points in the chromatin structure. These rendezvous points are the dyad and the linker DNA between histones, positions in the chromatin which are more likely to be accessible by repair proteins engaged in a random search. The feasibility of this mechanism is tested by considering the statistical mechanics of DNA containing a single lesion wrapped onto the nucleosome. We consider lesions which make the DNA either more flexible or more rigid by modeling the lesion as either a decrease or an increase in the bending energy. We include this energy in a partition function model of nucleosome breathing. Our results indicate that the steady state for a breathing nucleosome will most likely position the lesion at the dyad or in the linker, depending on the energy of the lesion. A role for DNA binding proteins and chromatin remodelers is suggested based on their ability to alter the mechanical properties of the DNA and DNA-histone binding, respectively. We speculate that these positions around the nucleosome potentially serve as rendezvous points where DNA lesions may be encountered by repair proteins which may be sterically hindered from searching the rest of the nucleosomal DNA. The strength of the repositioning is strongly dependent on the structural details of the DNA lesion and the wrapping and breathing of the nucleosome. A more sophisticated evaluation of this proposed mechanism will require detailed information about breathing dynamics, the structure of partially wrapped nucleosomes, and the structural properties of damaged DNA.
Bunnik, Evelien M; Polishko, Anton; Prudhomme, Jacques; Ponts, Nadia; Gill, Sarjeet S; Lonardi, Stefano; Le Roch, Karine G
In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite. Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes. The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.
DNA sites wrapped into chromatin are sterically occluded from proteins that must bind for processes such as RNA transcription and DNA repair. However, the role of chromatin compaction in biological function is poorly understood. To understand the biological functions of chromatin compaction, we constructed nucleosome arrays that are built with a tandem repeat of high affinity nucleosome positioning sequences, which contain probes for DNA accessibility and chromatin structure. I will describe our results that use restriction enzyme digestion and fluorescence resonance energy transfer to determine the probability for DNA site exposure within compacted nucleosome arrays and the time scale for changes in chromatin compaction. I will then discuss how these results help explain how proteins gain access to DNA sites buried within chromatin.
Salih, F; Salih, B; Kogan, S; Trifonov, E N
Alu sequences carry periodical pattern with CG dinucleotides (CpG) repeating every 31-32 bases. Similar distances are observed in distribution of DNA curvature in crystallized nucleosomes, at positions +/-1.5 and +/-4.5 periods of DNA from nucleosome DNA dyad. Since CG elements are also found to impart to nucleosomes higher stability when positioned at +/-1.5 sites, it suggests that CG dinucleotides may play a role in modulation of the nucleosome strength when the CG elements are methylated. Thus, Alu sequences may harbor special epigenetic nucleosomes with methylation-dependent regulatory functions. Nucleosome DNA sequence probe is suggested to detect locations of such regulatory nucleosomes in the sequences.
Pascuzzi, Pete E; Flores-Vergara, Miguel A; Lee, Tae-Jin; Sosinski, Bryon; Vaughn, Matthew W; Hanley-Bowdoin, Linda; Thompson, William F; Allen, George C
Scaffold or matrix attachment regions (S/MARs) are found in all eukaryotes. The pattern of distribution and genomic context of S/MARs is thought to be important for processes such as chromatin organization and modulation of gene expression. Despite the importance of such processes, much is unknown about the large-scale distribution and sequence content of S/MARs in vivo. Here, we report the use of tiling microarrays to map 1358 S/MARs on Arabidopsis thaliana chromosome 4 (chr4). S/MARs occur throughout chr4, spaced much more closely than in the large plant and animal genomes that have been studied to date. Arabidopsis S/MARs can be divided into five clusters based on their association with other genomic features, suggesting a diversity of functions. While some Arabidopsis S/MARs may define structural domains, most occur near the transcription start sites of genes. Genes associated with these S/MARs have an increased probability of expression, which is particularly pronounced in the case of transcription factor genes. Analysis of sequence motifs and 6-mer enrichment patterns show that S/MARs are preferentially enriched in poly(dA:dT) tracts, sequences that resist nucleosome formation, and the majority of S/MARs contain at least one nucleosome-depleted region. This global view of S/MARs provides a framework to begin evaluating genome-scale models for S/MAR function.
Chiquet, Matthias; Blumer, Susan; Angelini, Manuela; Mitsiadis, Thimios A.; Katsaros, Christos
During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis. PMID:27656150
Bintu, Lacramioara; Ishibashi, Toyotaka; Dangkulwanich, Manchuta; Wu, Yueh-Yi; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos
SUMMARY The nucleosome represents a mechanical barrier to transcription that operates as a general regulator of gene expression. We investigate how each nucleosomal component – the histone tails, the specific histone-DNA contacts, and the DNA sequence – contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones. In contrast, point mutations that affect histone-DNA contacts at the dyad abolish the barrier to transcription in the central region by decreasing the local wrapping rate. Moreover, we show that the nucleosome amplifies sequence-dependent transcriptional pausing, an effect mediated through the structure of the nascent RNA. Each of these nucleosomal elements controls transcription elongation by affecting distinctly the density and duration of polymerase pauses, thus providing multiple and alternative mechanisms for control of gene expression by chromatin remodeling and transcription factors. PMID:23141536
Bintu, Lacramioara; Kopaczynska, Marta; Hodges, Courtney; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos
Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process via the distribution of transcribed nucleosome positions. Significantly, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and that this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer, and histone-histone dissociation quantitatively rationalizes our observations and unifies results obtained with other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression. PMID:22081017
Pasi, Marco; Lavery, Richard
DNA loop formation on nucleosomes is strongly implicated in chromatin remodeling and occurs spontaneously in nucleosomes subjected to superhelical stress. The nature of such loops depends crucially on the balance between DNA deformation and DNA interaction with the nucleosome core. Currently, no high-resolution structural data on these loops exist. Although uniform rod models have been used to study loop size and shape, these models make assumptions concerning DNA mechanics and DNA–core binding. We present here atomic-scale molecular dynamics simulations for two different loop sizes. The results point to the key role of localized DNA kinking within the loops. Kinks enable the relaxation of DNA bending strain to be coupled with improved DNA–core interactions. Kinks lead to small, irregularly shaped loops that are asymmetrically positioned with respect to the nucleosome core. We also find that loop position can influence the dynamics of the DNA segments at the extremities of the nucleosome. PMID:27098037
Nucleosome Core Particle grown on STS-81. The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication. Principal Investigator's are Dr. Dan Carter and Dr. Gerard Bunick of New Century Pharmaceuticals.
Nucleosome Core Particle grown on STS-81. The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication. Principal Investigator's are Dr. Dan Carter and Dr. Gerard Bunick of New Century Pharmaceuticals.
Transcription initiation in vivo without classical transactivators: DNA kinks flanking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription.
Angermayr, Michaela; Oechsner, Ulrich; Gregor, Kerstin; Schroth, Gary P; Bandlow, Wolfhard
The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level. The promoter has been dissected in order to define elements that effect constitutive transcription. Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)(6). Nucleotide sequences 5' of this element only marginally affect transcription suggesting that promoter activation can dispense with transactivators and essentially involves basal transcription. We show that the core promoter of AKY2 is constitutively kept free of nucleosomes. Analyses of permutated AKY2 promoter DNA revealed the presence of bent DNA. DNA structure analysis by computer and by mutation identified two kinks flanking an interstitial stretch of 65 bp of moderately bent core promoter DNA. Kinked DNA is likely incompatible with packaging into nucleosomes and responsible for positioning nucleosomes at the flanks allowing unimpeded access of the basal transcription machinery to the core promoter. The data show that in yeast, constitutive gene expression can dispense with classical transcriptional activator proteins, if two prerequisites are met: (i) the core promoter is kept free of nucleosomes; this can be due to structural properties of the DNA as an alternative to chromatin remodeling factors; and (ii) the core promoter is pre-bent to allow a high rate of basal transcription initiation.
Introduction A postpartum diagnosis of breast cancer is an independent predictor of metastases, however the reason is unknown. In rodents, the window of postpartum mammary gland involution promotes tumor progression, suggesting a role for breast involution in the poor prognosis of human postpartum breast cancers. Rodent mammary gland involution is characterized by the programmed elimination of the secretory lobules laid down in preparation for lactation. This tissue involution process involves massive epithelial cell death, stromal remodeling, and immune cell infiltration with similarities to microenvironments present during wound healing and tumor progression. Here, we characterize breast tissue from premenopausal women with known reproductive histories to determine the extent, duration and cellular mechanisms of postpartum lobular involution in women. Methods Adjacent normal breast tissues from premenopausal women (n = 183) aged 20 to 45 years, grouped by reproductive categories of nulliparous, pregnant and lactating, and by time since last delivery were evaluated histologically and by special stain for lobular area, lobular type composition, apoptosis and immune cell infiltration using computer assisted quantitative methods. Results Human nulliparous glands were composed dominantly of small (approximately 10 acini per lobule) and medium (approximately 35 acini per lobule) sized lobules. With pregnancy and lactation, a >10 fold increase in breast epithelial area was observed compared to nulliparous cases, and lactating glands were dominated by mature lobules (>100 acini per lobule) with secretory morphology. Significant losses in mammary epithelial area and mature lobule phenotypes were observed within 12 months postpartum. By 18 months postpartum, lobular area content and lobule composition were indistinguishable from nulliparous cases, data consistent with postpartum involution facilitating regression of the secretory lobules developed in preparation for
Zhou, Xu; Blocker, Alexander W; Airoldi, Edoardo M; O'Shea, Erin K
Understanding chromatin function requires knowing the precise location of nucleosomes. MNase-seq methods have been widely applied to characterize nucleosome organization in vivo, but generally lack the accuracy to determine the precise nucleosome positions. Here we develop a computational approach leveraging digestion variability to determine nucleosome positions at a base-pair resolution from MNase-seq data. We generate a variability template as a simple error model for how MNase digestion affects the mapping of individual nucleosomes. Applied to both yeast and human cells, this analysis reveals that alternatively positioned nucleosomes are prevalent and create significant heterogeneity in a cell population. We show that the periodic occurrences of dinucleotide sequences relative to nucleosome dyads can be directly determined from genome-wide nucleosome positions from MNase-seq. Alternatively positioned nucleosomes near transcription start sites likely represent different states of promoter nucleosomes during transcription initiation. Our method can be applied to map nucleosome positions in diverse organisms at base-pair resolution. DOI: http://dx.doi.org/10.7554/eLife.16970.001 PMID:27623011
Miller, Thomas C. R.; Simon, Bernd; Rybin, Vladimir; Grötsch, Helga; Curtet, Sandrine; Khochbin, Saadi; Carlomagno, Teresa; Müller, Christoph W.
Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain–DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition. PMID:27991587
Itoh, Toshimasa; Fairall, Louise; Muskett, Frederick W.; Milano, Charles P.; Watson, Peter J.; Arnaudo, Nadia; Saleh, Almutasem; Millard, Christopher J.; El-Mezgueldi, Mohammed; Martino, Fabrizio; Schwabe, John W.R.
Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1. The amino-terminal region forms a tight dimerization domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxy-terminal domain of DNTTIP1 has a structure related to the SKI/SNO/DAC domain, despite lacking obvious sequence homology. We show that this domain in DNTTIP1 mediates interaction with both DNA and nucleosomes. Thus, DNTTIP1 acts as a dimeric chromatin binding module in the HDAC1:MIDEAS corepressor complex. PMID:25653165
Chung, Ho-Ryun; Vingron, Martin
Eukaryotic DNA is organized into a macromolecular structure called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of two copies of each of the four core histones and DNA. The nucleosomal organization and the positions of nucleosomes have profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is therefore of general interest. Among the many determinants of nucleosome positioning, the DNA sequence has been proposed to have a major role. Here, we analyzed more than 860,000 nucleosomal DNA sequences to identify sequence features that guide the formation of nucleosomes in vivo. We found that both a periodic enrichment of AT base pairs and an out-of-phase oscillating enrichment of GC base pairs as well as the overall preference for GC base pairs are determinants of nucleosome positioning. The preference for GC pairs can be related to a lower energetic cost required for deformation of the DNA to wrap around the histones. In line with this idea, we found that only incorporation of both signal components into a sequence model for nucleosome formation results in maximal predictive performance on a genome-wide scale. In this manner, one achieves greater predictive power than published approaches. Our results confirm the hypothesis that the DNA sequence has a major role in nucleosome positioning in vivo.
Chereji, Razvan; Morozov, Alexandre
Eukaryotic genomes are organized into arrays of nucleosomes, in which stretches of 147 base-pairs (bp) of DNA are wrapped around octameric histones. Recently, a new approach for direct mapping of nucleosome centers at bp resolution was developed [Brogaard et al., Nature 486, 496-501 (2012)] and some intriguing results appeared. About 40% of the inter-dyad distances are smaller than 147 bp, which imply massive nucleosome unwrapping, genome-wide, in vivo. The histogram of the inter-dyad distances presents small oscillations which indicate a step-wise unwrapping of the nucleosomal DNA from the histone. We present a statistical mechanics model for the nucleosome unwrapping, which is able to take into account sequence-dependent binding energies, sequence-independent potential barriers and wells, effective two-body interactions between the nucleosomes, competition between different species, cooperative-binding, and other important factors which dictate the nucleosome distribution along the DNA. We are able to reproduce the distribution of the inter-dyad distances, which cannot be obtained if there is no nucleosome unwrapping. The nucleosome unwrapping model can explain also the variable DNA accessibility and the nucleosome-induced cooperativity, which were observed experimentally.
Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins
Foulk, Michael S.; Urban, John M.; Casella, Cinzia; Gerbi, Susan A.
Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-exo digestion of nonreplicating genomic DNA (LexoG0) enriches GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand–independent λ-exo biases in NS-seq and validated this approach at the rDNA locus. The λ-exo–controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na+ instead of K+ in the λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq. PMID:25695952
Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.
Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.
Tsunaka, Yasuo; Fujiwara, Yoshie; Oyama, Takuji; Hirose, Susumu; Morikawa, Kosuke
Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.
Tsunaka, Yasuo; Fujiwara, Yoshie; Oyama, Takuji; Hirose, Susumu; Morikawa, Kosuke
Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT–histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3–H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid–H2A steric collision on the H2A-docking surface of the H3–H4 tetramer within the nucleosome induces H2A–H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone. PMID:26966247
Bryant, Gene O; Prabhu, Vidya; Floer, Monique; Wang, Xin; Spagna, Dan; Schreiber, David; Ptashne, Mark
The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome "remodeler" rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4's action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription-one that requires the action of SWI
Frenkel, Zakharia M; Trifonov, Edward N; Volkovich, Zeev; Bettecken, Thomas
This communication reports on the nucleosome positioning patterns (bendability matrices) for the human genome, derived from over 8_million nucleosome DNA sequences obtained from apoptotically digested lymphocytes. This digestion procedure is used here for the first time for the purpose of extraction and sequencing of the nucleosome DNA fragments. The dominant motifs suggested by the matrices of DNA bendability calculated for light and heavy isochores are significantly different. Both, however, are in full agreement with the linear description YRRRRRYYYYYR, and with earlier derivations by N-gram extensions. Thus, the choice of the nucleosome positioning patterns crucially depends on the G + C composition of the analyzed sequences.
Kiryanov, G I; Kintsurashvili, L N; Isaeva, L V; Zakharova, M G
It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.
Garcia, Jennifer F.; Dumesic, Phillip A.; Hartley, Paul D.; El-Samad, Hana; Madhani, Hiten D.
High-resolution nucleosome occupancy maps of heterochromatic regions of wild-type and silencing-defective mutants of the fission yeast Schizosaccharomyces pombe revealed that heterochromatin induces the elimination of nucleosome-free regions (NFRs). NFRs associated with transcription initiation sites as well as those not associated with promoters are affected. We dissected the roles of the histone H3K9 methyltransferase Clr4 and the HP1 proteins Swi6 and Chp2, as well as the two catalytic activities of the SHREC histone deacetylase (HDAC)/ATPase effector complex. Strikingly, different DNA sites have distinct combinatorial requirements for these factors: Five classes of NFRs were identified that are eliminated by silencing factors through a mechanistic hierarchy governed by Clr4. The SHREC HDAC activity plays a major role in the elimination of class I–IV NFRs by antagonizing the action of RSC, a remodeling complex implicated in NFR formation. We propose that heterochromatin formation involves the deployment in several sequence-specific mechanisms to eliminate gaps between nucleosomes, thereby blocking access to the DNA. PMID:20675407
Background Nucleosome distribution along chromatin dictates genomic DNA accessibility and thus profoundly influences gene expression. However, the underlying mechanism of nucleosome formation remains elusive. Here, taking a structural perspective, we systematically explored nucleosome formation potential of genomic sequences and the effect on chromatin organization and gene expression in S. cerevisiae. Results We analyzed twelve structural features related to flexibility, curvature and energy of DNA sequences. The results showed that some structural features such as DNA denaturation, DNA-bending stiffness, Stacking energy, Z-DNA, Propeller twist and free energy, were highly correlated with in vitro and in vivo nucleosome occupancy. Specifically, they can be classified into two classes, one positively and the other negatively correlated with nucleosome occupancy. These two kinds of structural features facilitated nucleosome binding in centromere regions and repressed nucleosome formation in the promoter regions of protein-coding genes to mediate transcriptional regulation. Based on these analyses, we integrated all twelve structural features in a model to predict more accurately nucleosome occupancy in vivo than the existing methods that mainly depend on sequence compositional features. Furthermore, we developed a novel approach, named DLaNe, that located nucleosomes by detecting peaks of structural profiles, and built a meta predictor to integrate information from different structural features. As a comparison, we also constructed a hidden Markov model (HMM) to locate nucleosomes based on the profiles of these structural features. The result showed that the meta DLaNe and HMM-based method performed better than the existing methods, demonstrating the power of these structural features in predicting nucleosome positions. Conclusions Our analysis revealed that DNA structures significantly contribute to nucleosome organization and influence chromatin structure and gene
Lewis, Daniel R; Olex, Amy L; Lundy, Stacey R; Turkett, William H; Fetrow, Jacquelyn S; Muday, Gloria K
To identify gene products that participate in auxin-dependent lateral root formation, a high temporal resolution, genome-wide transcript abundance analysis was performed with auxin-treated Arabidopsis thaliana roots. Data analysis identified 1246 transcripts that were consistently regulated by indole-3-acetic acid (IAA), partitioning into 60 clusters with distinct response kinetics. We identified rapidly induced clusters containing auxin-response functional annotations and clusters exhibiting delayed induction linked to cell division temporally correlated with lateral root induction. Several clusters were enriched with genes encoding proteins involved in cell wall modification, opening the possibility for understanding mechanistic details of cell structural changes that result in root formation following auxin treatment. Mutants with insertions in 72 genes annotated with a cell wall remodeling function were examined for alterations in IAA-regulated root growth and development. This reverse-genetic screen yielded eight mutants with root phenotypes. Detailed characterization of seedlings with mutations in cellulase3/glycosylhydrolase9b3 and leucine rich extensin2, genes not normally linked to auxin response, revealed defects in the early and late stages of lateral root development, respectively. The genes identified here using kinetic insight into expression changes lay the foundation for mechanistic understanding of auxin-mediated cell wall remodeling as an essential feature of lateral root development.
Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J
Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223
Rudolf, Martin; Seckerdieck, Katja; Grisanti, Salvatore; Curcio, Christine A
Drusen, the pathognomonic lesion of age-related macular degeneration, are dynamic and undergo both growth and regression. Using histochemistry to localise esterified cholesterol (EC), we investigated small drusen to discover signs of dynamism. Flat mounts of Bruch's membrane were prepared from peripheral retinas of six donor eyes without chorioretinal pathology that were preserved within 6 h of death. Tissues were pretreated with ethanol to extract native unesterified cholesterol, incubated with cholesterol esterase and stained with filipin to bind unesterified cholesterol that was newly released by hydrolysis. Tissues were imaged with wide-field epifluorescence microscopy. Diameters were measured and internal substructures (shells, lakes) assessed using previous descriptors. Of 676 drusen with mean diameter of 26.87 µm, 41.6% were stained homogeneously and 45.7% had lakes of pooled EC. Clusters of 2-7 drusen with similar staining patterns accounted for 25.3% of drusen. Increased EC content near the druse rim (shells) occurred in 10.5%. Over half of very small drusen at the edge of clinical detectability have evidence for internal remodelling, suggesting that both formative and removal events are present early in the druse lifecycle.
Castelnovo, Martin; Grauwin, Sébastian
We present in this article an original approach to compute the electrophoretic mobility of rigid nucleo-protein complexes like nucleosomes. This model allows us to address theoretically the influence of complex position along DNA, as well as wrapped length of DNA on the electrophoretic mobility of the complex. The predictions of the model are in qualitative agreement with experimental results on mononucleosomes assembled on short DNA fragments (<400 bp). Influences of additional experimental parameters like gel concentration, ionic strength, and effective charges are also discussed in the framework of the model, and are found to be qualitatively consistent with experiments when available. Based on the present model, we propose a simple semi-empirical formula describing positioning of nucleosomes as seen through electrophoresis. PMID:17277181
Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Sur, Sanjiv; Hegde, Muralidhar L.; Tian, Bing; Saavedra-Molina, Alfredo; Brasier, Allan R.; Ba, Xueqing; Boldogh, Istvan
Reactive oxygen species (ROS) generated by environmental exposures, and endogenously as by-products of respiration, oxidatively modify biomolecules including DNA. Accumulation of ROS-induced DNA damage has been implicated in various diseases that involve inflammatory processes, and efficient DNA repair is considered critical in preventing such diseases. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base-excision repair (OGG1-BER) pathway. Recent studies have shown that the OGG1-BER byproduct 8-oxoG base forms a complex with cytosolic OGG1, activating small GTPases and downstream cell signaling in cultured cells and lungs. This implies that persistent OGG1-BER could result in signaling leading to histological changes in airways. To test this, we mimicked OGG1-BER by repeatedly challenging airways with its repair product 8-oxoG base. Gene expression was analyzed by RNA sequencing (RNA-Seq) and qRT-PCR, and datasets were evaluated by gene ontology and statistical tools. RNA-Seq analysis identified 3252 differentially expressed transcripts (2435 up- and 817 downregulated, Z3-fold change). Among the upregulated transcripts, 2080 mRNAs were identified whose encoded protein products were involved in modulation of the actin family cytoskeleton, extracellular matrix, cell adhesion, cadherin, and cell junctions, affecting biological processes such as tissue development, cell-to-cell adhesion, cell communication, and the immune system. These data are supported by histological observations showing epithelial alterations, subepithelial fibrosis, and collagen deposits in the lungs. These data imply that continuous challenge by the environment and consequent OGG1-BER-driven signaling trigger gene expression consistent with airway remodeling. PMID:26187872
Lo, Stanley M; McElroy, Kyle A; Francis, Nicole J
Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity
Bouazoune, Karim; Mitterweger, Angelika; Längst, Gernot; Imhof, Axel; Akhtar, Asifa; Becker, Peter B.; Brehm, Alexander
Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi-2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to dHP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein–DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain-associated activity. PMID:12006495
Tambalo, Stefano; Peruzzotti-Jametti, Luca; Rigolio, Roberta; Fiorini, Silvia; Bontempi, Pietro; Mallucci, Giulia; Balzarotti, Beatrice; Marmiroli, Paola; Sbarbati, Andrea; Cavaletti, Guido
encephalomyelitis (EAE) represents a reliable model of the chronic-progressive variant of MS. fMRI studies in EAE have not been performed extensively up to now. This paper reports fMRI studies in a rat model of MS with somatosensory stimulation of the forepaw. We demonstrated modifications in the recruitment of cortical areas consistent with data from MS patients. To the best of our knowledge, this is the first report of cortical remodeling in a preclinical in vivo model of MS. PMID:26157006
Tao, Chenyu; Li, Juan; Chen, Baobao; Chi, Daming; Zeng, Yaqiong
The nucleosome is the basic structural unit of chromosomes, and its occupancy and distribution in promoters are crucial for the regulation of gene expression. During the growth process of porcine oocytes, the “growing” oocytes (SF) have a much higher transcriptional activity than the “fully grown” oocytes (BF). However, the chromosome status of the two kinds of oocytes remains poorly understood. In this study, we profiled the nucleosome distributions of SF and BF with as few as 1000 oocytes. By comparing the altered regions, we found that SF tended toward nucleosome loss and more open chromosome architecture than BF did. BF had decreased nucleosome occupancy in the coding region and increased nucleosome occupancy in the promoter compared to SF. The nucleosome occupancy of SF was higher than that of BF in the GC-poor regions, but lower than that of BF in the GC-rich regions. The nucleosome distribution around the transcriptional start site (TSS) of all the genes of the two samples was basically the same, but the nucleosome occupancy around the TSS of SF was lower than that of BF. GO functional annotation of genes with different nucleosome occupancy in promoter showed the genes were mainly involved in cell, cellular process, and metabolic process biological process. The results of this study revealed the dynamic reorganization of porcine oocytes in different developmental stages and the critical role of nucleosome arrangement during the oocyte growth process. PMID:28333987
Scovell, William M
High mobility group protein 1 (HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N’ and N’’) remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed (1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (2) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome. PMID:27247709
Wight, Andrew; Yang, Doo; Ioshikhes, Ilya; Makrigiannis, Andrew P
Beyond its role in genomic organization and compaction, the nucleosome is believed to participate in the regulation of gene transcription. Here, we report a computational method to evaluate the nucleosome sensitivity for a transcription factor over a given stretch of the genome. Sensitive factors are predicted to be those with binding sites preferentially contained within nucleosome boundaries and lacking 10 bp periodicity. Based on these criteria, the Acute Myeloid Leukemia-1a (AML-1a) transcription factor, a regulator of immune gene expression, was identified as potentially sensitive to nucleosomal regulation within the mouse Ly49 gene family. This result was confirmed in RMA, a cell line with natural expression of Ly49, using MNase-Seq to generate a nucleosome map of chromosome 6, where the Ly49 gene family is located. Analysis of this map revealed a specific depletion of nucleosomes at AML-1a binding sites in the expressed Ly49A when compared to the other, silent Ly49 genes. Our data suggest that nucleosome-based regulation contributes to the expression of Ly49 genes, and we propose that this method of predicting nucleosome sensitivity could aid in dissecting the regulatory role of nucleosomes in general.
Doiguchi, Masamichi; Nakagawa, Takeya; Imamura, Yuko; Yoneda, Mitsuhiro; Higashi, Miki; Kubota, Kazuishi; Yamashita, Satoshi; Asahara, Hiroshi; Iida, Midori; Fujii, Satoshi; Ikura, Tsuyoshi; Liu, Ziying; Nandu, Tulip; Kraus, W. Lee; Ueda, Hitoshi; Ito, Takashi
Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network. PMID:26888216
Datta, Kaberi; Basak, Trayambak; Varshney, Swati; Sengupta, Shantanu; Sarkar, Sagartirtha
Myocardial infarction is one of the leading causes of cardiac dysfunction, failure and sudden death. Post infarction cardiac remodeling presents a poor prognosis, with 30%-45% of patients developing heart failure, in a period of 5-25years. Oxidative stress has been labelled as the primary causative factor for cardiac damage during infarction, however, the impact it may have during the process of post infarction remodeling has not been well probed. In this study, we have implemented iTRAQ proteomics to catalogue proteins and functional processes, participating both temporally (early and late phases) and spatially (infarct and remote zones), during post myocardial infarction remodeling of the heart as functions of the differential oxidative stress manifest during the remodeling process. Cardiac metabolism was the dominant network to be affected during infarction and the remodeling time points considered in this study. A distinctive expression pattern of cytoskeletal proteins was also observed with increased remodeling time points. Further, it was found that the cytoskeletal protein Desmin, aggregated in the infarct zone during the remodeling process, mediated by the protease Calpain1. Taken together, all of these data in conjunction may lay the foundation to understand the effects of oxidative stress on the remodeling process and elaborate the mechanism behind the compromised cardiac function observed during post myocardial infarction remodeling.
Sandoz, Kelsi M; Popham, David L; Beare, Paul A; Sturdevant, Daniel E; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A
A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance.
Sandoz, Kelsi M.; Popham, David L.; Beare, Paul A.; Sturdevant, Daniel E.; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A.
A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3–3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3–3 cross-links as opposed to 16% 3–3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella’s environmental resistance. PMID:26909555
Boutinaud, Marion; Galio, Laurent; Lollivier, Vanessa; Finot, Laurence; Wiart, Sandra; Esquerré, Diane; Devinoy, Eve
Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.
Chevereau, G.; Palmeira, L.; Thermes, C.; Arneodo, A.; Vaillant, C.
The nucleosome ordering observed in vivo along yeast genes is described by a thermodynamical model of nonuniform fluid of 1D hard rods confined by two excluding energy barriers at gene extremities. For interbarrier distances L≲1.5kbp, nucleosomes equilibrate into a crystal-like configuration with a nucleosome repeat length (NRL) L/ñ165bp, where n is the number of regularly positioned nucleosomes. We also observe “bistable” genes with a fuzzy chromatin resulting from a statistical mixing of two crystal states, one with an expanded chromatin (NRL ˜L/n) and the other with a compact one (NRL ˜L/(n+1)). By means of single nucleosome switching, bistable genes may drastically alter their expression level as suggested by their higher transcriptional plasticity. These results enlighten the role of the intragenic chromatin on gene expression regulation.
Zaugg, Judith B.; Luscombe, Nicholas M.
Nucleosomes play an important role in gene regulation. Molecular studies observed that nucleosome binding in promoters tends to be repressive. In contrast, genomic studies have delivered conflicting results: An analysis of yeast grown on diverse carbon sources reported that nucleosome occupancies remain largely unchanged between conditions, whereas a study of the heat-shock response suggested that nucleosomes get evicted at promoters of genes with increased expression. Consequently, there are few general principles that capture the relationship between chromatin organization and transcriptional regulation. Here, we present a qualitative model for nucleosome positioning in Saccharomyces cerevisiae that helps explain important properties of gene expression. By integrating publicly available data sets, we observe that promoter-bound nucleosomes assume one of four discrete configurations that determine the active and silent transcriptional states of a gene, but not its expression level. In TATA-box-containing promoters, nucleosome architecture indicates the amount of transcriptional noise. We show that >20% of genes switch promoter states upon changes in cellular conditions. The data suggest that DNA-binding transcription factors together with chromatin-remodeling enzymes are primarily responsible for the nucleosome architecture. Our model for promoter nucleosome architecture reconciles genome-scale findings with molecular studies; in doing so, we establish principles for nucleosome positioning and gene expression that apply not only to individual genes, but across the entire genome. The study provides a stepping stone for future models of transcriptional regulation that encompass the intricate interplay between cis- and trans-acting factors, chromatin, and the core transcriptional machinery. PMID:21930892
Zraly, Claudia B; Dingwall, Andrew K
Nucleosome remodeling catalyzed by the ATP-dependent SWI/SNF complex is essential for regulated gene expression. Transcriptome profiling studies in flies and mammals identified cell cycle and hormone responsive genes as important targets of remodeling complex activities. Loss of chromatin remodeling function has been linked to developmental abnormalities and aggressive cancers. The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and conditional mutant alleles to identify unique and important functions of two conserved Brm complex core subunits, SNR1/SNF5 and BRM/SNF2-SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region. Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.
Hughes, Amanda L; Jin, Yi; Rando, Oliver J; Struhl, Kevin
Although the genomic pattern of nucleosome positioning is broadly conserved, quantitative aspects vary over evolutionary timescales. We identify the cis and trans determinants of nucleosome positioning using a functional evolutionary approach involving S. cerevisiae strains containing large genomic regions from other yeast species. In a foreign species, nucleosome depletion at promoters is maintained over poly(dA:dT) tracts, whereas internucleosome spacing and all other aspects of nucleosome positioning tested are not. Interestingly, the locations of the +1 nucleosome and RNA start sites shift in concert. Strikingly, in a foreign species, nucleosome-depleted regions occur fortuitously in coding regions, and they often act as promoters that are associated with a positioned nucleosome array linked to the length of the transcription unit. We suggest a three-step model in which nucleosome remodelers, general transcription factors, and the transcriptional elongation machinery are primarily involved in generating the nucleosome positioning pattern in vivo. Copyright © 2012 Elsevier Inc. All rights reserved.
Horlbeck, Max A; Witkowsky, Lea B; Guglielmi, Benjamin; Replogle, Joseph M; Gilbert, Luke A; Villalta, Jacqueline E; Torigoe, Sharon E; Tjian, Robert; Weissman, Jonathan S
The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties. DOI: http://dx.doi.org/10.7554/eLife.12677.001 PMID:26987018
Rapoport, Alexandra E; Trifonov, Edward N
Linguistic (word count) analysis of prokaryotic genome sequences, by Shannon N-gram extension, reveals that the dominant hidden motifs in A+T rich genomes are T(A)(T)A and G(A)(T)C with uncertain number of repeating A and T. Since prokaryotic sequences are largely protein-coding, the motifs would correspond to amphipathic alpha-helices with alternating lysine and phenylalanine as preferential polar and non-polar residues. The motifs are also known in eukaryotes, as nucleosome positioning patterns. Their existence in prokaryotes as well may serve for binding of histone-like proteins to DNA. In this case the above patterns in prokaryotes may be considered as "anticipated" nucleosome positioning patterns which, quite likely, existed in prokaryotic genomes before the evolutionary separation between eukaryotes and prokaryotes.
Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya
Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253
Nagy, Gergely; Ünnep, Renáta; Zsiros, Ottó; Tokutsu, Ryutaro; Takizawa, Kenji; Porcar, Lionel; Moyet, Lucas; Petroutsos, Dimitris; Garab, Győző; Finazzi, Giovanni; Minagawa, Jun
Plants respond to changes in light quality by regulating the absorption capacity of their photosystems. These short-term adaptations use redox-controlled, reversible phosphorylation of the light-harvesting complexes (LHCIIs) to regulate the relative absorption cross-section of the two photosystems (PSs), commonly referred to as state transitions. It is acknowledged that state transitions induce substantial reorganizations of the PSs. However, their consequences on the chloroplast structure are more controversial. Here, we investigate how state transitions affect the chloroplast structure and function using complementary approaches for the living cells of Chlamydomonas reinhardtii. Using small-angle neutron scattering, we found a strong periodicity of the thylakoids in state 1, with characteristic repeat distances of ∼200 Å, which was almost completely lost in state 2. As revealed by circular dichroism, changes in the thylakoid periodicity were paralleled by modifications in the long-range order arrangement of the photosynthetic complexes, which was reduced by ∼20% in state 2 compared with state 1, but was not abolished. Furthermore, absorption spectroscopy reveals that the enhancement of PSI antenna size during state 1 to state 2 transition (∼20%) is not commensurate to the decrease in PSII antenna size (∼70%), leading to the possibility that a large part of the phosphorylated LHCIIs do not bind to PSI, but instead form energetically quenched complexes, which were shown to be either associated with PSII supercomplexes or in a free form. Altogether these noninvasive in vivo approaches allow us to present a more likely scenario for state transitions that explains their molecular mechanism and physiological consequences. PMID:24639515
Schlingman, Daniel J; Mack, Andrew H; Kamenetska, Masha; Mochrie, Simon G J; Regan, Lynne
The dynamic packaging of DNA into chromatin is a key determinant of eukaryotic gene regulation and epigenetic inheritance. Nucleosomes are the basic unit of chromatin, and therefore the accessible states of the nucleosome must be the starting point for mechanistic models regarding these essential processes. Although the existence of different unwound nucleosome states has been hypothesized, there have been few studies of these states. The consequences of multiple states are far reaching. These states will behave differently in all aspects, including their interactions with chromatin remodelers, histone variant exchange, and kinetic properties. Here, we demonstrate the existence of two distinct states of the unwound nucleosome, which are accessible at physiological forces and ionic strengths. Using optical tweezers, we measure the rates of unwinding and rewinding for these two states and show that the rewinding rates from each state are different. In addition, we show that the probability of unwinding into each state is dependent on the applied force and ionic strength. Our results demonstrate not only that multiple unwound states exist but that their accessibility can be differentially perturbed, suggesting possible roles for these states in gene regulation. For example, different histone variants or modifications may facilitate or suppress access to DNA by promoting unwinding into one state or the other. We anticipate that the two unwound states reported here will be the basis for future models of eukaryotic transcriptional control.
Racki, Lisa R; Narlikar, Geeta J
ATP-dependent chromatin remodeling complexes enable rapid rearrangements in chromatin structure in response to developmental cues. The ATPase subunits of remodeling complexes share homology with the helicase motifs of DExx box helicases. Recent single-molecule experiments indicate that, like helicases, many of these complexes use ATP to translocate on DNA. Despite sharing this fundamental property, two key classes of remodeling complexes, the ISWI class and the SWI/SNF class, generate distinct remodeled products. SWI/SNF complexes generate nucleosomes with altered positions, nucleosomes with DNA loops and nucleosomes that are capable of exchanging histone dimers or octamers. In contrast, ISWI complexes generate nucleosomes with altered positions but in standard structures. Here, we draw analogies to monomeric and dimeric helicases and propose that ISWI and SWI/SNF complexes catalyze different outcomes in part because some ISWI complexes function as dimers while SWI/SNF complexes function as monomers.
Xiong, Jie; Gao, Shan; Dui, Wen; Yang, Wentao; Chen, Xiao; Taverna, Sean D.; Pearlman, Ronald E.; Ashlock, Wendy; Miao, Wei; Liu, Yifan
The ciliate protozoan Tetrahymena thermophila contains two types of structurally and functionally differentiated nuclei: the transcriptionally active somatic macronucleus (MAC) and the transcriptionally silent germ-line micronucleus (MIC). Here, we demonstrate that MAC features well-positioned nucleosomes downstream of transcription start sites and flanking splice sites. Transcription-associated trans-determinants promote nucleosome positioning in MAC. By contrast, nucleosomes in MIC are dramatically delocalized. Nucleosome occupancy in MAC and MIC are nonetheless highly correlated with each other, as well as with in vitro reconstitution and predictions based upon DNA sequence features, revealing unexpectedly strong contributions from cis-determinants. In particular, well-positioned nucleosomes are often matched with GC content oscillations. As many nucleosomes are coordinately accommodated by both cis- and trans-determinants, we propose that their distribution is shaped by the impact of these nucleosomes on the mutational and transcriptional landscape, and driven by evolutionary selection. PMID:27488188
Brown, Alice C N; Oddos, Stephane; Dobbie, Ian M; Alakoskela, Juha-Matti; Parton, Richard M; Eissmann, Philipp; Neil, Mark A A; Dunsby, Christopher; French, Paul M W; Davis, Ilan; Davis, Daniel M
Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.
Wenke, Jamie L.; McDonald, W. Hayes; Schey, Kevin L.
Purpose To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. Methods Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. Results Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. Conclusions Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ. PMID:27537260
Background Pathogens dependent upon vectors for transmission to new hosts undergo environment specific changes in gene transcription dependent on whether they are replicating in the vector or the mammalian host. Differential gene transcription, especially of potential vaccine candidates, is of interest in Anaplasma marginale, the tick-borne causative agent of bovine anaplasmosis. Methods RNA-seq technology allowed a comprehensive analysis of the transcriptional status of A. marginale genes in two conditions: bovine host blood and tick derived cell culture, a model for the tick vector. Quantitative PCR was used to assess transcription of a set of genes in A. marginale infected tick midguts and salivary glands at two time points during the transmission cycle. Results Genes belonging to fourteen pathways or component groups were found to be differentially transcribed in A. marginale in the bovine host versus the tick vector. One of the most significantly altered groups was composed of surface proteins. Of the 56 genes included in the surface protein group, eight were up regulated and 26 were down regulated. The down regulated surface protein encoding genes include several that are well studied due to their immunogenicity and function. Quantitative PCR of a set of genes demonstrated that transcription in tick cell culture most closely approximates transcription in salivary glands of recently infected ticks. Conclusions The ISE6 tick cell culture line is an acceptable model for early infection in tick salivary glands, and reveals disproportionate down regulation of surface protein genes in the tick. Transcriptional profiling in other cell lines may help us simulate additional microenvironments. Understanding vector-specific alteration of gene transcription, especially of surface protein encoding genes, may aid in the development of vaccines or transmission blocking therapies. PMID:24751137
Jones, B W; Kondo, M; Terasaki, H; Lin, Y; McCall, M; Marc, R E
Retinal photoreceptor degeneration takes many forms. Mutations in rhodopsin genes or disorders of the retinal pigment epithelium, defects in the adenosine triphosphate binding cassette transporter, ABCR gene defects, receptor tyrosine kinase defects, ciliopathies and transport defects, defects in both transducin and arrestin, defects in rod cyclic guanosine 3',5'-monophosphate phosphodiesterase, peripherin defects, defects in metabotropic glutamate receptors, synthetic enzymatic defects, defects in genes associated with signaling, and many more can all result in retinal degenerative disease like retinitis pigmentosa (RP) or RP-like disorders. Age-related macular degeneration (AMD) and AMD-like disorders are possibly due to a constellation of potential gene targets and gene/gene interactions, while other defects result in diabetic retinopathy or glaucoma. However, all of these insults as well as traumatic insults to the retina result in retinal remodeling. Retinal remodeling is a universal finding subsequent to retinal degenerative disease that results in deafferentation of the neural retina from photoreceptor input as downstream neuronal elements respond to loss of input with negative plasticity. This negative plasticity is not passive in the face of photoreceptor degeneration, with a phased revision of retinal structure and function found at the molecular, synaptic, cell, and tissue levels involving all cell classes in the retina, including neurons and glia. Retinal remodeling has direct implications for the rescue of vision loss through bionic or biological approaches, as circuit revision in the retina corrupts any potential surrogate photoreceptor input to a remnant neural retina. However, there are a number of potential opportunities for intervention that are revealed through the study of retinal remodeling, including therapies that are designed to slow down photoreceptor loss, interventions that are designed to limit or arrest remodeling events, and
Roca-Rivada, Arturo; Belen Bravo, Susana; Pérez-Sotelo, Diego; Alonso, Jana; Isabel Castro, Ana; Baamonde, Iván; Baltar, Javier; Casanueva, Felipe F.; Pardo, María
In the context of obesity, strong evidences support a distinctive pathological contribution of adipose tissue depending on its anatomical site of accumulation. Therefore, subcutaneous adipose tissue (SAT) has been lately considered metabolically benign compared to visceral fat (VAT), whose location is associated to the risk of developing cardiovascular disease, insulin resistance, and other associated comorbidities. Under the above situation, the chronic local inflammation that characterizes obese adipose tissue, has acquired a major role on the pathogenesis of obesity. In this work, we have analyzed for the first time human obese VAT and SAT secretomes using an improved quantitative proteomic approach for the study of tissue secretomes, Comparison of Isotope-Labeled Amino acid Incorporation Rates (CILAIR). The use of double isotope-labeling-CILAIR approach to analyze VAT and SAT secretomes allowed the identification of location-specific secreted proteins and its differential secretion. Additionally to the very high percentage of identified proteins previously implicated in obesity or in its comorbidities, this approach was revealed as a useful tool for the study of the obese adipose tissue microenvironment including extracellular matrix (ECM) remodeling and inflammatory status. The results herein presented reinforce the fact that VAT and SAT depots have distinct features and contribute differentially to metabolic disease. PMID:26198096
Butryn, Agata; Schuller, Jan M; Stoehr, Gabriele; Runge-Wollmann, Petra; Förster, Friedrich; Auble, David T; Hopfner, Karl-Peter
Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex. DOI: http://dx.doi.org/10.7554/eLife.07432.001 PMID:26258880
Paton, A.E.; Wilkinson-Singley, E.; Olins, D.W.
Nucleosome core particles form well defined complexes with the nuclear nonhistone proteins HMG 14 or 17. The binding of HMG 14 or 17 to nucleosomes results in greater stability of the nucleosomal DNA as shown by circular dichroism and thermal denaturation. Under appropriate conditions the binding is cooperative, and cooperativity is ionic strength dependent. The specificity and cooperative transitions of high mobility group (HMG) binding are preserved in 1 M urea. Specificity is lost in 4 M urea. Thermal denaturation and circular dichroism show a dramatic reversal of the effects of urea on nucleosomes when HMG 14 or 17 is bound, indicating stabilization of the nucleosome by HMG proteins. Complexes formed between reconstructed nucleosomes containing purified inner histones plus poly (dA-dT) and HMG 14 or 17 demonstrate that the HMG binding site requires only DNA and histones. Electron microscopy reveals no major structural alterations in the nucleosome upon binding of HMG 14 or 17. Cross-linking the nucleosome extensively with formaldehyde under cooperative HMG binding conditions does not prevent the ionic strength-dependent shift to noncooperative binding. This suggests mechanisms other than internal nucleosome conformational changes may be involved in cooperative HMG binding.
McAnena, Peter; Brown, James A. L.; Kerin, Michael J.
Traditionally the stratification of many cancers involves combining tumour and clinicopathological features (e.g., patient age; tumour size, grade, receptor status and location) to inform treatment options and predict recurrence risk and survival. However, current biomarkers often require invasive excision of the tumour for profiling, do not allow monitoring of the response to treatment and stratify patients into broad heterogeneous groups leading to inconsistent treatment responses. Here we explore and describe the benefits of using circulating biomarkers (nucleosomes and/or modifications to nucleosomes) as a non-invasive method for detecting cancer and monitoring response to treatment. Nucleosomes (DNA wound around eight core histone proteins) are responsible for compacting our genome and their composition and post-translational modifications are responsible for regulating gene expression. Here, we focus on breast and colorectal cancer as examples where utilizing circulating nucleosomes as biomarkers hold real potential as liquid biopsies. Utilizing circulating nucleosomes as biomarkers is an exciting new area of research that promises to allow both the early detection of cancer and monitoring of treatment response. Nucleosome-based biomarkers combine with current biomarkers, increasing both specificity and sensitivity of current tests and have the potential to provide individualised precision-medicine based treatments for patients. PMID:28075351
Al-Ani, Gada; Briggs, Koan; Malik, Shuja Shafi; Conner, Michael; Azuma, Yoshiaki; Fischer, Christopher J
The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β1 = 1.3 ± 0.6 nM, and 1/β2 = 13 ± 7 nM(2). Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.
Brennan, Lucy D.; Forties, Robert A.; Patel, Smita S.; Wang, Michelle D.
Proper cell function requires preservation of the spatial organization of chromatin modifications. Maintenance of this epigenetic landscape necessitates the transfer of parental nucleosomes to newly replicated DNA, a process that is stringently regulated and intrinsically linked to replication fork dynamics. This creates a formidable setting from which to isolate the central mechanism of transfer. Here we utilized a minimal experimental system to track the fate of a single nucleosome following its displacement, and examined whether DNA mechanics itself, in the absence of any chaperones or assembly factors, may serve as a platform for the transfer process. We found that the nucleosome is passively transferred to available dsDNA as predicted by a simple physical model of DNA loop formation. These results demonstrate a fundamental role for DNA mechanics in mediating nucleosome transfer and preserving epigenetic integrity during replication. PMID:27808093
Flores, Oscar; Deniz, Özgen; Soler-López, Montserrat; Orozco, Modesto
Nucleosome organization plays a key role in the regulation of gene expression. However, despite the striking advances in the accuracy of nucleosome maps, there are still severe discrepancies on individual nucleosome positioning and how this influences gene regulation. The variability among nucleosome maps, which precludes the fine analysis of nucleosome positioning, might emerge from diverse sources. We have carefully inspected the extrinsic factors that may induce diversity by the comparison of microccocal nuclease (MNase)-Seq derived nucleosome maps generated under distinct conditions. Furthermore, we have also explored the variation originated from intrinsic nucleosome dynamics by generating additional maps derived from cell cycle synchronized and asynchronous yeast cultures. Taken together, our study has enabled us to measure the effect of noise in nucleosome occupancy and positioning and provides insights into the underlying determinants. Furthermore, we present a systematic approach that may guide the standardization of MNase-Seq experiments in order to generate reproducible genome-wide nucleosome patterns. PMID:24586063
Yang, Yue; Yamada, Tomoko; Hill, Kelly K; Hemberg, Martin; Reddy, Naveen C; Cho, Ha Y; Guthrie, Arden N; Oldenborg, Anna; Heiney, Shane A; Ohmae, Shogo; Medina, Javier F; Holy, Timothy E; Bonni, Azad
Activity-dependent transcription influences neuronal connectivity, but the roles and mechanisms of inactivation of activity-dependent genes have remained poorly understood. Genome-wide analyses in the mouse cerebellum revealed that the nucleosome remodeling and deacetylase (NuRD) complex deposits the histone variant H2A.z at promoters of activity-dependent genes, thereby triggering their inactivation. Purification of translating messenger RNAs from synchronously developing granule neurons (Sync-TRAP) showed that conditional knockout of the core NuRD subunit Chd4 impairs inactivation of activity-dependent genes when neurons undergo dendrite pruning. Chd4 knockout or expression of NuRD-regulated activity genes impairs dendrite pruning. Imaging of behaving mice revealed hyperresponsivity of granule neurons to sensorimotor stimuli upon Chd4 knockout. Our findings define an epigenetic mechanism that inactivates activity-dependent transcription and regulates dendrite patterning and sensorimotor encoding in the brain. Copyright © 2016, American Association for the Advancement of Science.
Getun, Irina V.; Wu, Zhen K.; Bois, Philippe R.J.
Meiotic double strand breaks (DSBs) occur at discrete regions in the genome coined hotspots. Precisely what directs site selection of these DSBs is hotly debated and in particular it is unclear which chromatin features, and regulatory factors are necessary for a genomic region to initiate and resolve DSBs as a crossover (CO) event. In human and mouse, one layer of hotspot selection control is a recognition sequence element present at these sites that is bound by the Prdm9 zinc-finger protein. Furthermore, an overall open chromatin structure is thought to be required to allow access of the recombination machinery, and this is often dictated by the packaging of DNA around nucleosomes. We recently defined the nucleosome occupancy maps of four mouse recombination hotspots throughout meiosis. These analyses revealed no obvious dynamic changes in nucleosome occupancy, suggesting an intrinsic nature of recombinogenic sites, yet they also revealed that nucleosomes define zones of exclusion for CO resolution. Here, we discuss new evidence implicating nucleosome occupancy in recombinogenic repair and its potential roles in controlling chromatin structure at mouse meiotic hotspots. PMID:22572955
Cannan, Wendy J.; Tsang, Betty P.; Wallace, Susan S.; Pederson, David S.
Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. PMID:24891506
Liu, Zhonghua; Li, Fudong; Zhang, Beibei; Li, Sai; Wu, Jihui; Shi, Yunyu
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.
Zhao, Z; Xu, J; Chen, J; Kim, S; Reimers, M; Bacanu, S-A; Yu, H; Liu, C; Sun, J; Wang, Q; Jia, P; Xu, F; Zhang, Y; Kendler, K S; Peng, Z; Chen, X
Schizophrenia (SCZ) and bipolar disorder (BPD) are severe mental disorders with high heritability. Clinicians have long noticed the similarities of clinic symptoms between these disorders. In recent years, accumulating evidence indicates some shared genetic liabilities. However, what is shared remains elusive. In this study, we conducted whole transcriptome analysis of post-mortem brain tissues (cingulate cortex) from SCZ, BPD and control subjects, and identified differentially expressed genes in these disorders. We found 105 and 153 genes differentially expressed in SCZ and BPD, respectively. By comparing the t-test scores, we found that many of the genes differentially expressed in SCZ and BPD are concordant in their expression level (q⩽0.01, 53 genes; q⩽0.05, 213 genes; q⩽0.1, 885 genes). Using genome-wide association data from the Psychiatric Genomics Consortium, we found that these differentially and concordantly expressed genes were enriched in association signals for both SCZ (P<10(-7)) and BPD (P=0.029). To our knowledge, this is the first time that a substantially large number of genes show concordant expression and association for both SCZ and BPD. Pathway analyses of these genes indicated that they are involved in the lysosome, Fc gamma receptor-mediated phagocytosis, regulation of actin cytoskeleton pathways, along with several cancer pathways. Functional analyses of these genes revealed an interconnected pathway network centered on lysosomal function and the regulation of actin cytoskeleton. These pathways and their interacting network were principally confirmed by an independent transcriptome sequencing data set of the hippocampus. Dysregulation of lysosomal function and cytoskeleton remodeling has direct impacts on endocytosis, phagocytosis, exocytosis, vesicle trafficking, neuronal maturation and migration, neurite outgrowth and synaptic density and plasticity, and different aspects of these processes have been implicated in SCZ and BPD.
Winter, Deborah R.; Song, Lingyun; Mukherjee, Sayan; Furey, Terrence S.; Crawford, Gregory E.
DNase-seq is primarily used to identify nucleosome-depleted DNase I hypersensitive (DHS) sites genome-wide that correspond to active regulatory elements. However, ∼40 yr ago it was demonstrated that DNase I also digests with a ∼10-bp periodicity around nucleosomes matching the exposure of the DNA minor groove as it wraps around histones. Here, we use DNase-seq data from 49 samples representing diverse cell types to reveal this digestion pattern at individual loci and predict genomic locations where nucleosome rotational positioning, the orientation of DNA with respect to the histone surface, is stably maintained. We call these regions DNase I annotated regions of nucleosome stability (DARNS). Compared to MNase-seq experiments, we show DARNS correspond well to annotated nucleosomes. Interestingly, many DARNS are positioned over only one side of annotated nucleosomes, suggesting that the periodic digestion pattern attenuates over the nucleosome dyad. DARNS reproduce the arrangement of nucleosomes around transcription start sites and are depleted at ubiquitous DHS sites. We also generated DARNS from multiple lymphoblast cell line (LCL) samples. We found that LCL DARNS were enriched at DHS sites present in most of the original 49 samples but absent in LCLs, while multi-cell-type DARNS were enriched at LCL-specific DHS sites. This indicates that variably open DHS sites are often occupied by rotationally stable nucleosomes in cell types where the DHS site is closed. DARNS provide additional information about precise DNA orientation within individual nucleosomes not available from other nucleosome positioning assays and contribute to understanding the role of chromatin in gene regulation. PMID:23657885
Winter, Deborah R; Song, Lingyun; Mukherjee, Sayan; Furey, Terrence S; Crawford, Gregory E
DNase-seq is primarily used to identify nucleosome-depleted DNase I hypersensitive (DHS) sites genome-wide that correspond to active regulatory elements. However, ≈ 40 yr ago it was demonstrated that DNase I also digests with a ≈ 10-bp periodicity around nucleosomes matching the exposure of the DNA minor groove as it wraps around histones. Here, we use DNase-seq data from 49 samples representing diverse cell types to reveal this digestion pattern at individual loci and predict genomic locations where nucleosome rotational positioning, the orientation of DNA with respect to the histone surface, is stably maintained. We call these regions DNase I annotated regions of nucleosome stability (DARNS). Compared to MNase-seq experiments, we show DARNS correspond well to annotated nucleosomes. Interestingly, many DARNS are positioned over only one side of annotated nucleosomes, suggesting that the periodic digestion pattern attenuates over the nucleosome dyad. DARNS reproduce the arrangement of nucleosomes around transcription start sites and are depleted at ubiquitous DHS sites. We also generated DARNS from multiple lymphoblast cell line (LCL) samples. We found that LCL DARNS were enriched at DHS sites present in most of the original 49 samples but absent in LCLs, while multi-cell-type DARNS were enriched at LCL-specific DHS sites. This indicates that variably open DHS sites are often occupied by rotationally stable nucleosomes in cell types where the DHS site is closed. DARNS provide additional information about precise DNA orientation within individual nucleosomes not available from other nucleosome positioning assays and contribute to understanding the role of chromatin in gene regulation.
Dieker, Jürgen; Schlumberger, Wolfgang; McHugh, Neil; Hamann, Philip; van der Vlag, Johan; Berden, Jo H
Autoantibodies against nucleosomes are considered a hallmark of systemic lupus erythematosus (SLE). We compared in patients with proliferative lupus nephritis the diagnostic usefulness of a dsDNA-loaded nucleosome ELISA (anti-dsDNA-NcX) with ELISAs in which dsDNA or nucleosomes alone were coated. First, we analysed whether DNA loading on nucleosomes led to masking of epitopes by using defined monoclonal anti-DNA, anti-histone and nucleosome-specific autoantibodies to evaluate the accessibility of nucleosomal epitopes in the anti-dsDNA-NcX ELISA. Second, autoantibody levels were measured in these 3 ELISAs in 100 patients with proliferative lupus nephritis (LN) before immunosuppressive treatment and in 128 non-SLE disease controls. In patients with LN inter-assay comparisons and associations with clinical and serological parameters were analysed. The panel of monoclonal antibodies revealed that all epitopes were equally accessible in the anti-dsDNA-NcX ELISA as in the two other ELISAs. Patients with proliferative lupus nephritis were positive with dsDNA-loaded nucleosomes in 86%, with DNA in 66% and with nucleosomes in 85%. In the non-lupus disease control group these frequencies were 1.6% (2 out of 128) for both the anti-dsDNA-NcX and the anti-dsDNA ELISA and 0% in the anti-nucleosome ELISA. The levels in the anti-dsDNA-NcX ELISA were high in a group of patients with LN that showed absent reactivity in the anti-DNA or low levels in the anti-nucleosome ELISA. Anti-dsDNA-NcX positivity was associated with higher SLEDAI scores within this group. Within nucleosome-based ELISAs, we propose the anti-dsDNA-NcX ELISA as the preferred test system.
Spakowitz, Andrew; Sudhanshu, Bariz
The fundamental unit of packaged DNA, the nucleosome core particle, contains 146 base pairs of DNA wrapped 1.7 times around a cationic protein complex called the histone octamer. A string of nucleosomes is organized into higher-order structures at several hierarchical levels to form chromatin, a remarkable complex that is compact yet maintains accessibility for gene expression. We develop a theoretical model of the nucleosome core particle in order to extract detailed quantitative information from single-molecule measurements of a single nucleosome under tension. We employ the wormlike chain model to describe the DNA strand as a thermally fluctuating polymer chain. The chain adsorbs on a spool that represents the histone octamer. This model is directly compared to single-molecule experiments conducted in Carlos Bustamante's lab; we find good agreement between our theory and the experimental data. Our model reveals the mechanism that underlies structural transitions that are apparent in the experimental measurements and predicts the conditions where these transitions occur. We proceed to construct a free energy surface to predict the dynamic response in a single-molecule experiment with a time-dependent rate of unwinding the nucleosome. The combination of single-molecule experiments and our theoretical modeling gives detailed information about the specific interactions between DNA and histone proteins.
Sheinman, Michael; Chung, Ho-Ryun
Positioning of nucleosomes along a eukaryotic genome plays an important role in its organization and regulation. There are many different factors affecting the location of nucleosomes. Some can be viewed as preferential binding of a single nucleosome to different locations along the DNA and some as interactions between neighboring nucleosomes. In this study, we analyze positioning of nucleosomes and derive conditions for their good positioning. Using analytic and numerical approaches we find that, if the binding preferences are very weak, an interplay between the interactions and the binding preferences is essential for a good positioning of nucleosomes, especially on correlated energy landscapes. Analyzing the empirical energy landscape, we conclude that good positioning of nucleosomes in vivo is possible only if they strongly interact. In this case, our model, predicting long-length-scale fluctuations of nucleosomes' occupancy along the DNA, accounts well for the empirical observations.
Sheinman, Michael; Chung, Ho-Ryun
Positioning of nucleosomes along a eukaryotic genome plays an important role in its organization and regulation. There are many different factors affecting the location of nucleosomes. Some can be viewed as preferential binding of a single nucleosome to different locations along the DNA and some as interactions between neighboring nucleosomes. In this study, we analyze positioning of nucleosomes and derive conditions for their good positioning. Using analytic and numerical approaches we find that, if the binding preferences are very weak, an interplay between the interactions and the binding preferences is essential for a good positioning of nucleosomes, especially on correlated energy landscapes. Analyzing the empirical energy landscape, we conclude that good positioning of nucleosomes in vivo is possible only if they strongly interact. In this case, our model, predicting long-length-scale fluctuations of nucleosomes' occupancy along the DNA, accounts well for the empirical observations.
Howell, Steven; Jimenez-Useche, Isabel; Andresen, Kurt; Yuan, Chongli; Qiu, Xiangyun
Chromatin conformation and dynamics is central to gene functions including packaging, regulation, and repair. At the molecular level, the basic building block of chromatin is a nucleosome core particle (NCP) made of 147 base pairs (bp) of dsDNA wrapped around an octamer of histone proteins. These NCPs are connected by short 10-90 bps of linker DNA as beads on a string. Key factors determining the packaging of NCP arrays to form chromatin include ionic condition, linker DNA length, and epigenetic modifications, especially of the histone tails. We have investigated how the conformations of model tetra-NCP arrays are modulated by these factors using small angle x-ray scattering (SAXS). Here we present recent studies of the effects of ion (KCl and MgCl2), linker length, and histone modification (tail deletions) on NCP arrays. Our SAXS measurement makes it possible to learn about both the global compaction of NCP arrays and local inter-NCP spatial correlations within the same array.
Erdel, Fabian; Rippe, Karsten
The specific location of nucleosomes on DNA has important inhibitory or activating roles in the regulation of DNA-dependent processes as it affects the DNA accessibility. Nucleosome positions depend on the ATP-coupled activity of chromatin-remodelling complexes that translocate nucleosomes or evict them from the DNA. The mammalian cell harbors numerous different remodelling complexes that possess distinct activities. These can translate a variety of signals into certain patterns of nucleosome positions with specific functions. Although chromatin remodellers have been extensively studied in vitro, much less is known about how they operate in their cellular environment. Here, we review the cellular activities of the mammalian imitation switch proteins and discuss mechanisms by which they are targeted to sites where their activity is needed. © 2011 The Authors Journal compilation © 2011 FEBS.
Langecker, Martin; Ivankin, Andrey; Carson, Spencer; Kinney, Shannon R M; Simmel, Friedrich C; Wanunu, Meni
Nucleosomes are the fundamental repeating units of chromatin, and dynamic regulation of their positioning along DNA governs gene accessibility in eukaryotes. Although epigenetic factors have been shown to influence nucleosome structure and dynamics, the impact of DNA methylation on nucleosome packaging remains controversial. Further, all measurements to date have been carried out under zero-force conditions. In this paper, we present the first automated force measurements that probe the impact of CpG DNA methylation on nucleosome stability. In solid-state nanopore force spectroscopy, a nucleosomal DNA tail is captured into a pore and pulled on with a time-varying electrophoretic force until unraveling is detected. This is automatically repeated for hundreds of nucleosomes, yielding statistics of nucleosome lifetime vs electrophoretic force. The force geometry, which is similar to displacement forces exerted by DNA polymerases and helicases, reveals that nucleosome stability is sensitive to DNA sequence yet insensitive to CpG methylation. Our label-free method provides high-throughput data that favorably compares with other force spectroscopy experiments and is suitable for studying a variety of DNA-protein complexes.
Gansen, Alexander; Hieb, Aaron R; Böhm, Vera; Tóth, Katalin; Langowski, Jörg
Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.
Shinkai, Soya; Nozaki, Tadasu; Maeshima, Kazuhiro
The mammalian genome is organized into submegabase-sized chromatin domains (CDs) including topologically associating domains, which have been identified using chromosome conformation capture-based methods. Single-nucleosome imaging in living mammalian cells has revealed subdiffusively dynamic nucleosome movement. It is unclear how single nucleosomes within CDs fluctuate and how the CD structure reflects the nucleosome movement. Here, we present a polymer model wherein CDs are characterized by fractal dimensions and the nucleosome fibers fluctuate in a viscoelastic medium with memory. We analytically show that the mean-squared displacement (MSD) of nucleosome fluctuations within CDs is subdiffusive. The diffusion coefficient and the subdiffusive exponent depend on the structural information of CDs. This analytical result enabled us to extract information from the single-nucleosome imaging data for HeLa cells. Our observation that the MSD is lower at the nuclear periphery region than the interior region indicates that CDs in the heterochromatin-rich nuclear periphery region are more compact than those in the euchromatin-rich interior region with respect to the fractal dimensions as well as the size. Finally, we evaluated that the average size of CDs is in the range of 100–500 nm and that the relaxation time of nucleosome movement within CDs is a few seconds. Our results provide physical and dynamic insights into the genome architecture in living cells. PMID:27764097
Sugiyama, Masaaki; Arimura, Yasuhiro; Shirayama, Kazuyoshi; Fujita, Risa; Oba, Yojiro; Sato, Nobuhiro; Inoue, Rintaro; Oda, Takashi; Sato, Mamoru; Heenan, Richard K; Kurumizaka, Hitoshi
Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.
Yang, Xi; Yan, Hong
Based on the anisotropic deformation of DNA structure in nucleosomes, the non-linear dimensionality reduction algorithm Isomap is used to derive a structural signal accounting for most structural variances from the DNA structural data of nucleosome crystals. The analysis of this structural signal by continuous wavelet transform has successfully revealed the common regularity of nucleosome DNA deformation and also the peculiarity of structural configurations in nucleosomes with particular histone or DNA composition, or special ligands. The relationship between the constituent dinucleotides and the signal energy distribution shows that the fluctuation of a structural signal is sensitive to certain dinucleotide types.
Eaton, Matthew L.; Galani, Kyriaki; Kang, Sukhyun; Bell, Stephen P.; MacAlpine, David M.
The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. PMID:20351051
Buttgereit, Andreas; Weber, Cornelia; Garbe, Christoph S; Friedrich, Oliver
Duchenne muscular dystrophy (DMD) is a common inherited muscle disease showing chronic inflammation and progressive muscle weakness. Absent dystrophin renders sarcolemma more Ca(2+) -permeable, disturbs signalling and triggers inflammation. Sustained degeneration/regeneration cycles render muscle cytoarchitecture susceptible to remodelling. Quantitative morphometry was introduced in living cells using second-harmonic generation (SHG) microscopy of myosin. As the time course of cellular remodelling is not known, we used SHG microscopy in mdx muscle fibres over a wide age range for three-dimensional (3D) rendering and detection of verniers and cosine angle sums (CASs). Wild-type (wt) and transgenic mini-dystrophin mice (MinD) were also studied. Vernier densities (VDs) declined in wt and MinD fibres until adulthood, while in mdx fibres, VDs remained significantly elevated during the life span. CAS values were close to unity in adult wt and MinD fibres, in agreement with tight regular myofibril orientation, while always smaller in mdx fibres. Using SHG 3D morphometry, we identified two types of altered ultrastructure: branched fibres and a novel, previously undetected 'chaotic' fibre type, both of which can be classified by distinct CAS and VD combinations. We present a novel model of tissue remodelling in dystrophic progression with age that involves the transition from normal to chaotic to branched fibres. Our model predicts a ~50% contribution of altered cytoarchitecture to progressive force loss with age. We also provide an improved automated image algorithm that is suitable for future ageing studies in human myopathies.
Lee, Junwoo; Choi, Eun Shik; Lee, Daeyoup
The ability of elongating RNA polymerase II (RNAPII) to regulate the nucleosome barrier is poorly understood because we do not know enough about the involved factors and we lack a conceptual framework to model this process. Our group recently identified the conserved Fun30/SMARCAD1 family chromatin-remodeling factor, Fun30(Fft3), as being critical for relieving the nucleosome barrier during RNAPII-mediated elongation, and proposed a model illustrating how Fun30(Fft3) may contribute to nucleosome disassembly during RNAPII-mediated elongation. Here, we present a model that describes nucleosome dynamics during RNAPII-mediated elongation in mathematical terms and addresses the involvement of Fun30(Fft3) in this process.
Chodavarapu, Ramakrishna K.; Feng, Suhua; Bernatavichute, Yana V.; Chen, Pao-Yang; Stroud, Hume; Yu, Yanchun; Hetzel, Jonathan; Kuo, Frank; Kim, Jin; Cokus, Shawn J.; Casero, David; Bernal, Maria; Huijser, Peter; Clark, Amander T.; Krämer, Ute; Merchant, Sabeeha S.; Zhang, Xiaoyu; Jacobsen, Steven E.; Pellegrini, Matteo
Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer1, 2. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana utilizing massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified ten base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results suggest that nucleosome positioning strongly influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA suggesting that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA Pol II was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition. PMID:20512117
Riffo-Campos, Ángela L; Castillo, Josefa; Tur, Gema; González-Figueroa, Paula; Georgieva, Elena I; Rodríguez, José L; López-Rodas, Gerardo; Rodrigo, M Isabel; Franco, Luis
Histone post-translational modifications and nucleosome remodeling are coordinate events involved in eukaryotic transcriptional regulation. There are relatively few data on the time course with which these events occur in individual nucleosomes. As a contribution to fill this gap, we first describe the nature and time course of structural changes in the nucleosomes -2, -1, and +1 of the murine Egr1 gene upon induction. To initiate the transient activation of the gene, we used the stimulation of MLP29 cells with phorbol esters and the in vivo activation after partial hepatectomy. In both models, nucleosomes -1 and +1 are partially evicted, whereas nucleosomes +1 and -2 slide downstream during transcription. The sliding of the latter nucleosome allows the EGR1 protein to bind its site, resulting in the repression of the gene. To decide whether EGR1 is involved in the sliding of nucleosome -2, Egr1 was knocked down. In the absence of detectable EGR1, the nucleosome still slides and remains downstream longer than in control cells, suggesting that the product of the gene may be rather involved in the returning of the nucleosome to the basal position. Moreover, the presence of eight epigenetic histone marks has been determined at a mononucleosomal level in that chromatin region. H3S10phK14ac, H3K4me3, H3K9me3, and H3K27me3 are characteristic of nucleosome +1, and H3K9ac and H4K16ac are mainly found in nucleosome -1, and H3K27ac predominates in nucleosomes -2 and -1. The temporal changes in these marks suggest distinct functions for some of them, although changes in H3K4me3 may result from histone turnover.
Levendosky, Robert F; Sabantsev, Anton; Deindl, Sebastian; Bowman, Gregory D
Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo. DOI: http://dx.doi.org/10.7554/eLife.21356.001 PMID:28032848
Carey, Michael; Li, Bing; Workman, Jerry L.
Summary The coordinated action of histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling enzymes in promoter-dependent transcription initiation represents a paradigm for how epigenetic information regulates gene expression. However, little is known about how such enzymes function during transcription elongation. Here we investigated the role of RSC, a bromodomain-containing ATPase, in nucleosome transcription in vitro. Purified S. cerevisiae RNA polymerase II (pol II) arrests at two primary locations on a positioned mononucleosome. RSC stimulates passage of pol II through these sites. The function of RSC in elongation requires the energy of ATP hydrolysis. Moreover, the SAGA and NuA4 HATs strongly stimulated RSC’s effect on elongation. The stimulation correlates closely with Acetyl-CoA-dependent recruitment of RSC to nucleosomes. Thus, RSC can recognize acetylated nucleosomes and facilitate passage of pol II through them. These data support the view that histone modifications regulate accessibility of the coding region to pol II. PMID:17081996
Retinoblastoma protein (pRB) interacts with E2F and other protein factors to play a pivotal role in regulating the expression of target genes that induce cell cycle arrest, apoptosis, and differentiation. pRB controls the local promoter activity and has the ability to change the structure of nucleosomes and/or chromosomes via histone modification, epigenetic changes, chromatin remodeling, and chromosome organization. Functional inactivation of pRB perturbs these cellular events and causes dysregulated cell growth and chromosome instability, which are hallmarks of cancer cells. The role of pRB in regulation of nucleosome/chromatin structures has been shown to link to tumor suppression. This review focuses on the ability of pRB to control nucleosome/chromatin structures via physical interactions with histone modifiers and chromatin factors and describes cancer therapies based on targeting these protein factors.
Retinoblastoma protein (pRB) interacts with E2F and other protein factors to play a pivotal role in regulating the expression of target genes that induce cell cycle arrest, apoptosis, and differentiation. pRB controls the local promoter activity and has the ability to change the structure of nucleosomes and/or chromosomes via histone modification, epigenetic changes, chromatin remodeling, and chromosome organization. Functional inactivation of pRB perturbs these cellular events and causes dysregulated cell growth and chromosome instability, which are hallmarks of cancer cells. The role of pRB in regulation of nucleosome/chromatin structures has been shown to link to tumor suppression. This review focuses on the ability of pRB to control nucleosome/chromatin structures via physical interactions with histone modifiers and chromatin factors and describes cancer therapies based on targeting these protein factors. PMID:28101510
Pan, Qian; Li, Min; Shi, Yao-Long; Liu, Huwei; Speakman, John R; Wang, De-Hua
Mongolian gerbils (Meriones unguiculatus) have high physiological flexibility in response to acute temperature changes, and have the widest thermoneutral zone (TNZ, 26.5-38.9 °C) reported among small mammals. At the upper critical temperature (T(uc), 38.9 °C), body temperatures of gerbils were significantly increased (39-41 °C) while metabolic rates were maintained at the basal level. In contrast, below the lower critical temperature (T(lc), 26.5 °C), metabolism was elevated and body temperature stable. Rapid changes in mitochondrial membrane lipidome were hypothesized to play an important role during acute thermoregulation of gerbils. Taking advantage of a recent lipidomic technique, we examined changes in the membrane phospholipids environment and free fatty acids (FFA) production in mitochondria between 38 and 27 °C (in the TNZ), and between 27 and 16 °C (below the TNZ). At 38 °C, acute heat stress elicited distinct remodeling in mitochondrial membrane lipidome which related to a potential decrease in mitochondrial respiration and membrane fluidity compared to 27 °C. At 16 °C, a sharply decreased unsaturation index and increased chain lengths were detected in mitochondrial FFA production both in muscle and brown adipose tissue. Our results suggest that mitochondrial membrane lipid remodeling may stabilize membrane function and activity of respiration related membrane protein to maintain a stable metabolic rate at T(uc), and improve heat production by decomposing less fluid fatty acid conjugates of membrane lipids under acute cold exposure. These data therefore imply an important role of membrane remodeling during acute thermoregulation in a non-hibernating endotherm.
Zhang, Tao; Zhang, Wenli; Jiang, Jiming
The fundamental unit of chromatin is the nucleosome that consists of a protein octamer composed of the four core histones (Hs; H3, H4, H2A, and H2B) wrapped by 147 bp of DNA. Nucleosome occupancy and positioning have proven to be dynamic and have a critical impact on expression, regulation, and evolution of eukaryotic genes. We developed nucleosome occupancy and positioning data sets using leaf tissue of rice (Oryza sativa) and both leaf and flower tissues of Arabidopsis (Arabidopsis thaliana). We show that model plant and animal species share the fundamental characteristics associated with nucleosome dynamics. Only 12% and 16% of the Arabidopsis and rice genomes, respectively, were occupied by well-positioned nucleosomes. The cores of positioned nucleosomes were enriched with G/C dinucleotides and showed a lower C→T mutation rate than the linker sequences. We discovered that nucleosomes associated with heterochromatic regions were more spaced with longer linkers than those in euchromatic regions in both plant species. Surprisingly, different nucleosome densities were found to be associated with chromatin in leaf and flower tissues in Arabidopsis. We show that deep MNase-seq data sets can be used to map nucleosome occupancy of specific genomic loci and reveal gene expression patterns correlated with chromatin dynamics in plant genomes. © 2015 American Society of Plant Biologists. All Rights Reserved.
Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto
Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity.
Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto
Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620
Cui, Feng; Zhurkin, Victor B.
Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken β-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the ‘601’ nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp — an accuracy exceeding that of earlier predictions. Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning ‘rules’ they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL ±3.5 and ±5.5, which is similar to the α-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the ‘601’ clone
Cui, Feng; Zhurkin, Victor B
Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken beta-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the '601' nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp - an accuracy exceeding that of earlier predictions. Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning 'rules' they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL+/- 3.5 and +/- 5.5, which is similar to the alpha-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the '601' clone) the
Shen, Xiaoyan; Wang, Zenglan; Song, Xiaofeng; Xu, Jiajia; Jiang, Chunyun; Zhao, Yanxiu; Ma, Changle; Zhang, Hui
Plants can successfully improve their resistance to previously lethal salinity stress by a short exposure to low levels of salt stress, a process known as salt acclimation (SA). In spite of its fundamental significance in theoretical study and agricultural practice, the molecular mechanisms underlying plant SA remain elusive. In this study, we found that salt acclimated Arabidopsis young seedlings can survive subsequent 200 mM NaCl stress. RNA-seq was performed to analyze the genome-wide transcriptional response under SA conditions. Among 518 differentially expressed genes (DEGs) under SA, 366 up-regulated genes were enriched for cell wall biosynthesis, osmoregulation, oxidative stress, or transcription factors. Seven DEGs participate in the synthesis of lignin and 24 DEGs encode plant cell wall proteins, suggesting the importance of cell wall remodeling under SA. Furthermore, in comparison to non-acclimated salt stress, 228 of 245 DEGs were repressed by acclimated salt stress, including many genes related to ethylene biosynthesis and signaling pathway. In addition, MAPK6, a major component of the ethylene signaling pathway, was found to play a crucial role in SA. Our transcriptomic analysis has provided important insight on the roles of transcription factors, cell wall remodeling, and the ethylene biosynthesis and signaling pathways during SA in Arabidopsis.
Good, Paul D.; Kendall, Ann; Ignatz-Hoover, James; Miller, Erin L.; Pai, Dave A.; Rivera, Sara R.; Carrick, Brian; Engelke, David R.
Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed. PMID:23707796
Good, Paul D; Kendall, Ann; Ignatz-Hoover, James; Miller, Erin L; Pai, Dave A; Rivera, Sara R; Carrick, Brian; Engelke, David R
Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.
Labonne, Jonathan D J; Dorweiler, Jane E; McGinnis, Karen M
Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.
Labonne, Jonathan D. J.; Dorweiler, Jane E.; McGinnis, Karen M.
Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1–1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1–1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression. PMID:23538550
Yarrington, Robert M; Goodrum, Jenna M; Stillman, David J
Nucleosome-depleted regions (NDRs) are present immediately adjacent to the transcription start site in most eukaryotic promoters. Here we show that NDRs in the upstream promoter region can profoundly affect gene regulation. Chromatin at the yeast HO promoter is highly repressive and numerous coactivators are required for expression. We modified the HO promoter with segments from the well-studied CLN2 NDR, creating chimeric promoters differing in nucleosome occupancy but with binding sites for the same activator, SBF. Nucleosome depletion resulted in substantial increases in both factor binding and gene expression and allowed activation from a much longer distance, probably by allowing recruited coactivators to act further downstream. Nucleosome depletion also affected sequential activation of the HO promoter; HO activation typically requires the ordered recruitment of activators first to URS1, second to the left-half of URS2 (URS2-L), and finally to the right-half of URS2 (URS2-R), with each region representing distinct gates that must be unlocked to achieve activation. The absence of nucleosomes at URS2-L resulted in promoters no longer requiring both the URS1 and URS2-L gates, as either gate alone is now sufficient to promote binding of the SBF factor to URS2-R. Furthermore, nucleosome depletion at URS2 altered the timing of HO expression and bypassed the regulation that restricts expression to mother cells. Our results reveal insight into how nucleosomes can create a requirement for ordered recruitment of factors to facilitate complex transcriptional regulation.
Pachov, Georgi V.; Gabdoulline, Razif R.; Wade, Rebecca C.
Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber. PMID:21355036
Shaw, P A; Sahasrabuddhe, C G; Hodo, H G; Saunders, G F
Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes. PMID:693325
Geng, Liguo; Zhu, Meng; Wang, Yuzhuo; Cheng, Yang; Liu, Jia; Shen, Wei; Li, Zhihua; Zhang, Jiahui; Wang, Cheng; Jin, Guangfu; Ma, Hongxia; Shen, Hongbing; Hu, Zhibin; Dai, Juncheng
Chromatin remodeling complexes utilize the energy of ATP hydrolysis to remodel nucleosomes and have essential roles in transcriptional modulation. Increasing evidences indicate that these complexes directly interact with numerous proteins and regulate the formation of cancer. However, few studies reported the association of polymorphisms in chromatin remodeling genes and lung cancer. We hypothesized that variants in critical genes of chromatin remodeling pathway might contribute to the susceptibility of lung cancer. To validate this hypothesis, we systematically screened 40 polymorphisms in six key chromatin remodeling genes (SMARCA5, SMARCC2, SMARCD2, ARID1A, NR3C1 and SATB1) and evaluated them with a case-control study including 1341 cases and 1982 controls. Logistic regression revealed that four variants in NR3C1 and SATB1 were significantly associated with lung cancer risk after false discovery rate (FDR) correction [For NR3C1, rs9324921: odds ratio (OR)=1.23, P for FDR=0.029; rs12521436: OR=0.85, P for FDR=0.040; rs4912913: OR=1.17, P for FDR=0.040; For SATB1, rs6808523: OR=1.33, P for FDR=0.040]. Combing analysis presented a significant allele-dosage tendency for the number of risk alleles and lung cancer risk (Ptrend<0.001). Moreover, expression quantitative trait loci (eQTL) analysis revealed that these two genes were differently expressed between lung tumor and adjacent normal tissues in the database of The Cancer Genome Atlas (TCGA) (P=0.009 for rs6808523). These findings suggested that genetic variants in key chromatin remodeling genes may contribute to lung cancer risk in Chinese population. Further large and well-designed studies are warranted to validate our results.
Li, Bing; Pattenden, Samantha G.; Lee, Daeyoup; Gutiérrez, José; Chen, Jie; Seidel, Chris; Gerton, Jennifer; Workman, Jerry L.
The yeast histone variant H2AZ (Htz1) is implicated in transcription activation, prevention of the ectopic spread of heterochromatin, and genome integrity. Our genome-wide localization analysis revealed that Htz1 is widely, but nonrandomly, distributed throughout the genome in an SWR1-dependent manner. We found that Htz1 is enriched in intergenic regions compared with coding regions. Its occupancy is inversely proportional to transcription rates and the enrichment of the RNA polymerase II under different growth conditions. However, Htz1 does not seem to directly regulate transcription repression genome-wide; instead, the presence of Htz1 under the inactivated condition is essential for optimal activation of a subset of genes. In addition, Htz1 is not generally responsible for nucleosome positioning, even at those promoters where Htz1 is highly enriched. Finally, using a biochemical approach, we demonstrate that incorporation of Htz1 into nucleosomes inhibits activities of histone modifiers associated with transcription, Dot1, Set2, and NuA4 and reduces the nucleosome mobilization driven by chromatin remodeling complexes. These lines of evidence collectively suggest that Htz1 may serve to mark quiescent promoters for proper activation. PMID:16344463
Eukaryotic genomes encode an additional layer of genetic information, superimposed on top of the regulatory and coding information, that controls the organization of the genomic DNA into arrays of nucleosomes. We have developed a partial ability to read this nucleosome positioning code and predict the in vivo locations of nucleosomes. Our results suggest that genomes utilize the nucleosome positioning code to facilitate specific chromosome functions including to delineate functional versus nonfunctional binding sites for key gene regulatory proteins, and to define the next higher level of chromosome structure itself.
Collings, Clayton K; Waddell, Peter J; Anderson, John N
Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.
Malouf, Gabriel G; Su, Xiaoping; Yao, Hui; Gao, Jianjun; Xiong, Liangwen; He, Qiuming; Compérat, Eva; Couturier, Jérôme; Molinié, Vincent; Escudier, Bernard; Camparo, Philippe; Doss, Denaha J; Thompson, Erika J; Khayat, David; Wood, Christopher G; Yu, Willie; Teh, Bin T; Weinstein, John; Tannir, Nizar M
MITF/TFE translocation renal cell carcinoma (TRCC) is a rare subtype of kidney cancer. Its incidence and the genome-wide characterization of its genetic origin have not been fully elucidated. We performed RNA and exome sequencing on an exploratory set of TRCC (n = 7), and validated our findings using The Cancer Genome Atlas (TCGA) clear-cell RCC (ccRCC) dataset (n = 460). Using the TCGA dataset, we identified seven TRCC (1.5%) cases and determined their genomic profile. We discovered three novel partners of MITF/TFE (LUC7L3, KHSRP, and KHDRBS2) that are involved in RNA splicing. TRCC displayed a unique gene expression signature as compared with other RCC types, and showed activation of MITF, the transforming growth factor β1 and the PI3K complex targets. Genes differentially spliced between TRCC and other RCC types were enriched for MITF and ID2 targets. Exome sequencing of TRCC revealed a distinct mutational spectrum as compared with ccRCC, with frequent mutations in chromatin-remodeling genes (six of eight cases, three of which were from the TCGA). In two cases, we identified mutations in INO80D, an ATP-dependent chromatin-remodeling gene, previously shown to control the amplitude of the S phase. Knockdown of INO80D decreased cell proliferation in a novel cell line bearing LUC7L3-TFE3 translocation. This genome-wide study defines the incidence of TRCC within a ccRCC-directed project and expands the genomic spectrum of TRCC by identifying novel MITF/TFE partners involved in RNA splicing and frequent mutations in chromatin-remodeling genes. ©2014 American Association for Cancer Research.
Creamer, Kevin M.; Job, Godwin; Shanker, Sreenath; Neale, Geoffrey A.; Lin, Yuan-chi; Bartholomew, Blaine
Mit1 is the putative chromatin remodeling subunit of the fission yeast Snf2/histone deacetylase (HDAC) repressor complex (SHREC) and is known to repress transcription at regions of heterochromatin. However, how Mit1 modifies chromatin to silence transcription is largely unknown. Here we report that Mit1 mobilizes histone octamers in vitro and requires ATP hydrolysis and conserved chromatin tethering domains, including a previously unrecognized chromodomain, to remodel nucleosomes and silence transcription. Loss of Mit1 remodeling activity results in nucleosome depletion at specific DNA sequences that display low intrinsic affinity for the histone octamer, but its contribution to antagonizing RNA polymerase II (Pol II) access and transcription is not restricted to these sites. Genetic epistasis analyses demonstrate that SHREC subunits and the transcription-coupled Set2 histone methyltransferase, which is involved in suppression of cryptic transcription at actively transcribed regions, cooperate to silence heterochromatic transcripts. In addition, we have demonstrated that Mit1's remodeling activity contributes to SHREC function independently of Clr3's histone deacetylase activity on histone H3 K14. We propose that Mit1 is a chromatin remodeling factor that cooperates with the Clr3 histone deacetylase of SHREC and other chromatin modifiers to stabilize heterochromatin structure and to prevent access to the transcriptional machinery. PMID:24662054
Shaytan, Alexey K; Landsman, David; Panchenko, Anna R
Nucleosome variability is essential for their functions in compacting the chromatin structure and regulation of transcription, replication and cell reprogramming. The DNA molecule in nucleosomes is wrapped around an octamer composed of four types of core histones (H3, H4, H2A, H2B). Nucleosomes represent dynamic entities and may change their conformation, stability and binding properties by employing different sets of histone variants or by becoming post-translationally modified. There are many variants of histones H2A and H2B. Specific H2A and H2B variants may preferentially associate with each other resulting in different combinations of variants and leading to the increased combinatorial complexity of nucleosomes. In addition, the H2A-H2B dimer can be recognized and substituted by chaperones/remodelers as a distinct unit, can assemble independently and is stable during nucleosome unwinding. In this review we discuss how sequence and structural variations in H2A-H2B dimers may provide necessary complexity and confer the nucleosome functional variability.
The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β1 = 1.3 ± 0.6 nM, and 1/β2 = 13 ± 7 nM2. Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP. PMID:24898734
Osakabe, Akihisa; Adachi, Fumiya; Arimura, Yasuhiro; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi
DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.
Joshi, Sachindra R; Sarpong, Yaw C; Peterson, Ronald C; Scovell, William M
High mobility group protein 1 (HMGB1) interacts with DNA and chromatin to influence the regulation of transcription, DNA repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, ATP-independent manner. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N' and N″) remain stable and exhibit characteristics distinctly different from the canonical nucleosome. These findings complement previous studies that showed (i) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (ii) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that one aspect of the mechanism of HMGB1 action involves a restructuring of the nucleosome that appears to relax structural constraints within the nucleosome.
van der Heijden, Thijn; van Vugt, Joke J F A; Logie, Colin; van Noort, John
Nucleosome positioning dictates eukaryotic DNA compaction and access. To predict nucleosome positions in a statistical mechanics model, we exploited the knowledge that nucleosomes favor DNA sequences with specific periodically occurring dinucleotides. Our model is the first to capture both dyad position within a few base pairs, and free binding energy within 2 k(B)T, for all the known nucleosome positioning sequences. By applying Percus's equation to the derived energy landscape, we isolate sequence effects on genome-wide nucleosome occupancy from other factors that may influence nucleosome positioning. For both in vitro and in vivo systems, three parameters suffice to predict nucleosome occupancy with correlation coefficients of respectively 0.74 and 0.66. As predicted, we find the largest deviations in vivo around transcription start sites. This relatively simple algorithm can be used to guide future studies on the influence of DNA sequence on chromatin organization.
Workman, J L; Buchman, A R
The in vivo packaging of DNA with histone proteins to form chromatin makes its transcription a difficult process. Biochemical and genetic studies are beginning to reveal mechanistic details of how transcriptional regulatory factors confront at least two hurdles created by nucleosomes, the primary structural unit of chromatin. Regulatory factors must gain access to their respective binding sites and activate the formation of transcription complexes at core promoter elements. Distinct regulatory factors may be specialized to perform these functions.
Huebert, Dana J.; Kuan, Pei-Fen; Keleş, Sündüz
The response to stressful stimuli requires rapid, precise, and dynamic gene expression changes that must be coordinated across the genome. To gain insight into the temporal ordering of genome reorganization, we investigated dynamic relationships between changing nucleosome occupancy, transcription factor binding, and gene expression in Saccharomyces cerevisiae yeast responding to oxidative stress. We applied deep sequencing to nucleosomal DNA at six time points before and after hydrogen peroxide treatment and revealed many distinct dynamic patterns of nucleosome gain and loss. The timing of nucleosome repositioning was not predictive of the dynamics of downstream gene expression change but instead was linked to nucleosome position relative to transcription start sites and specific cis-regulatory elements. We measured genome-wide binding of the stress-activated transcription factor Msn2p over time and found that Msn2p binds different loci with different dynamics. Nucleosome eviction from Msn2p binding sites was common across the genome; however, we show that, contrary to expectation, nucleosome loss occurred after Msn2p binding and in fact required Msn2p. This negates the prevailing model that nucleosomes obscuring Msn2p sites regulate DNA access and must be lost before Msn2p can bind DNA. Together, these results highlight the complexities of stress-dependent chromatin changes and their effects on gene expression. PMID:22354995
Funke, Jonas J; Ketterer, Philip; Lieleg, Corinna; Korber, Philipp; Dietz, Hendrik
We establish a DNA origami based tool for quantifying conformational equilibria of biomolecular assemblies as a function of environmental conditions. As first application, we employed the tool to study the salt-induced disassembly of nucleosome core particles. To extract binding constants and energetic penalties, we integrated nucleosomes in the spectrometer such that unwrapping of the nucleosomal template DNA, leading from bent to more extended states was directly coupled to the conformation of the spectrometer. Nucleosome unwrapping was induced by increasing the ionic strength. The corresponding shifts in conformation equilibrium of the spectrometer were followed by direct conformation imaging using negative staining TEM and by FRET read out after gel electrophoretic separation of conformations. We find nucleosome dissociation constants in the picomolar range at low ionic strength (11 mM MgCl2), in the nanomolar range at intermediate ionic strength (11 mM MgCl2 with 0.5-1 M NaCl) and in the micromolar range at larger ionic strength (11 mM MgCl2 with ≥1.5 M NaCl). Integration of up to four nucleosomes stacked side-by-side, as it might occur within chromatin fibers, did not appear to affect the salt-induced unwrapping of nucleosomes. Presumably, such stacking interactions are already effectively screened at the nucleosome unwrapping conditions. Our spectrometer provides a modular platform with a direct read out to study conformational equilibria for targets from small biomolecules up to large macromolecular assemblies.
Chen, Wei; Luo, Liaofu; Zhang, Lirong
The occupancy of nucleosomes along chromosome is a key factor for gene regulation. However, except promoter regions, genome-wide properties and functions of nucleosome organization remain unclear in mammalian genomes. Using the computational model of Increment of Diversity with Quadratic Discriminant (IDQD) trained from the microarray data, the nucleosome occupancy score (NOScore) was defined and applied to splice junction regions of constitutive, cassette exon, alternative 3′ and 5′ splicing events in the human genome. We found an interesting relation between NOScore and RNA splicing: exon regions have higher NOScores compared with their flanking intron sequences in both constitutive and alternative splicing events, indicating the stronger nucleosome occupation potential of exon regions. In addition, NOScore valleys present at ∼25 bp upstream of the acceptor site in all splicing events. By defining folding diversity-to-energy ratio to describe RNA structural flexibility, we demonstrated that primary RNA transcripts from nucleosome occupancy regions are relatively rigid and those from nucleosome depleted regions are relatively flexible. The negative correlation between nucleosome occupation/depletion of DNA sequence and structural flexibility/rigidity of its primary transcript around splice junctions may provide clues to the deeper understanding of the unexpected role for nucleosome organization in the regulation of RNA splicing. PMID:20097656
Berná, Luisa; Chiribao, Maria Laura; Greif, Gonzalo; Rodriguez, Matias; Alvarez-Valin, Fernando; Robello, Carlos
American trypanosomiasis is a chronic and endemic disease which affects millions of people. Trypanosoma cruzi, its causative agent, has a life cycle that involves complex morphological and functional transitions, as well as a variety of environmental conditions. This requires a tight regulation of gene expression, which is achieved mainly by post-transcriptional regulation. In this work we conducted an RNAseq analysis of the three major life cycle stages of T. cruzi: amastigotes, epimastigotes and trypomastigotes. This analysis allowed us to delineate specific transcriptomic profiling for each stage, and also to identify those biological processes of major relevance in each state. Stage specific expression profiling evidenced the plasticity of T. cruzi to adapt quickly to different conditions, with particular focus on membrane remodeling and metabolic shifts along the life cycle. Epimastigotes, which replicate in the gut of insect vectors, showed higher expression of genes related to energy metabolism, mainly Krebs cycle, respiratory chain and oxidative phosphorylation related genes, and anabolism related genes associated to nucleotide and steroid biosynthesis; also, a general down-regulation of surface glycoprotein coding genes was seen at this stage. Trypomastigotes, living extracellularly in the bloodstream of mammals, express a plethora of surface proteins and signaling genes involved in invasion and evasion of immune response. Amastigotes mostly express membrane transporters and genes involved in regulation of cell cycle, and also express a specific subset of surface glycoprotein coding genes. In addition, these results allowed us to improve the annotation of the Dm28c genome, identifying new ORFs and set the stage for construction of networks of co-expression, which can give clues about coded proteins of unknown functions.
Greif, Gonzalo; Rodriguez, Matias; Alvarez-Valin, Fernando
American trypanosomiasis is a chronic and endemic disease which affects millions of people. Trypanosoma cruzi, its causative agent, has a life cycle that involves complex morphological and functional transitions, as well as a variety of environmental conditions. This requires a tight regulation of gene expression, which is achieved mainly by post-transcriptional regulation. In this work we conducted an RNAseq analysis of the three major life cycle stages of T. cruzi: amastigotes, epimastigotes and trypomastigotes. This analysis allowed us to delineate specific transcriptomic profiling for each stage, and also to identify those biological processes of major relevance in each state. Stage specific expression profiling evidenced the plasticity of T. cruzi to adapt quickly to different conditions, with particular focus on membrane remodeling and metabolic shifts along the life cycle. Epimastigotes, which replicate in the gut of insect vectors, showed higher expression of genes related to energy metabolism, mainly Krebs cycle, respiratory chain and oxidative phosphorylation related genes, and anabolism related genes associated to nucleotide and steroid biosynthesis; also, a general down-regulation of surface glycoprotein coding genes was seen at this stage. Trypomastigotes, living extracellularly in the bloodstream of mammals, express a plethora of surface proteins and signaling genes involved in invasion and evasion of immune response. Amastigotes mostly express membrane transporters and genes involved in regulation of cell cycle, and also express a specific subset of surface glycoprotein coding genes. In addition, these results allowed us to improve the annotation of the Dm28c genome, identifying new ORFs and set the stage for construction of networks of co-expression, which can give clues about coded proteins of unknown functions. PMID:28286708
Min, Hae-Ki; Sookoian, Silvia; Pirola, Carlos J.; Cheng, Jianfeng; Mirshahi, Faridoddin
PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling. PMID:24763554
Pinet, Florence; Cuvelliez, Marie; Kelder, Thomas; Amouyel, Philippe; Radonjic, Marijana; Bauters, Christophe
To elucidate the time-resolved molecular events underlying the LV remodeling (LVR) process, we developed a large-scale network model that integrates the 24 molecular variables (plasma proteins and non-coding RNAs) collected in the REVE-2 study at four time points (baseline, 1month, 3months and 1year) after MI. The REVE-2 network model was built by extending the set of REVE-2 variables with their mechanistic context based on known molecular interactions (1310 nodes and 8639 edges). Changes in the molecular variables between the group of patients with high LVR (>20%) and low LVR (<20%) were used to identify active network modules within the clusters associated with progression of LVR, enabling assessment of time-resolved molecular changes. Although the majority of molecular changes occur at the baseline, two network modules specifically show an increasing number of active molecules throughout the post-MI follow up: one involved in muscle filament sliding, containing the major troponin forms and tropomyosin proteins, and the other associated with extracellular matrix disassembly, including matrix metalloproteinases, tissue inhibitors of metalloproteinases and laminin proteins. For the first time, integrative network analysis of molecular variables collected in REVE-2 patients with known molecular interactions allows insight into time-dependent mechanisms associated with LVR following MI, linking specific processes with LV structure alteration. In addition, the REVE-2 network model provides a shortlist of prioritized putative novel biomarker candidates for detection of LVR after MI event associated with a high risk of heart failure and is a valuable resource for further hypothesis generation.
Failler, Marion; Corna, Stefano; Racagni, Giorgio; Mathé, Aleksander A.; Popoli, Maurizio
Background: Major depression is a severe mental illness that causes heavy social and economic burdens worldwide. A number of studies have shown that interaction between individual genetic vulnerability and environmental risk factors, such as stress, is crucial in psychiatric pathophysiology. In particular, the experience of stressful events in childhood, such as neglect, abuse, or parental loss, was found to increase the risk for development of depression in adult life. Here, to reproduce the gene x environment interaction, we employed an animal model that combines genetic vulnerability with early-life stress. Methods: The Flinders Sensitive Line rats (FSL), a validated genetic animal model of depression, and the Flinders Resistant Line (FRL) rats, their controls, were subjected to a standard protocol of maternal separation (MS) from postnatal days 2 to 14. A basal comparison between the two lines for the outcome of the environmental manipulation was performed at postnatal day 73, when the rats were into adulthood. We carried out a global proteomic analysis of purified synaptic terminals (synaptosomes), in order to study a subcellular compartment enriched in proteins involved in synaptic function. Two-dimensional gel electrophoresis (2-DE), mass spectrometry, and bioinformatic analysis were used to analyze proteins and related functional networks that were modulated by genetic susceptibility (FSL vs. FRL) or by exposure to early-life stress (FRL + MS vs. FRL and FSL + MS vs. FSL). Results: We found that, at a synaptic level, mainly proteins and molecular pathways related to energy metabolism and cellular remodeling were dysregulated. Conclusions: The present results, in line with previous works, suggest that dysfunction of energy metabolism and cytoskeleton dynamics at a synaptic level could be features of stress-related pathologies, in particular major depression. PMID:25522407
Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael
Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.
Cutter, Amber; Hayes, Jeffrey J.
The nucleosomal subunit organization of chromatin provides a multitude of functions. Nucleosomes elicit an initial ~7-fold linear compaction of genomic DNA. They provide a critical mechanism for stable repression of genes and other DNA-dependent activities by restricting binding of trans-acting factors to cognate DNA sequences. Conversely they are engineered to be nearly meta-stable and disassembled (and reassembled) in a facile manner to allow rapid access to the underlying DNA during processes such as transcription, replication and DNA repair. Nucleosomes protect the genome from DNA damaging agents and provide a lattice onto which a myriad of epigenetic signals are deposited. Moreover, vast strings of nucleosomes provide a framework for assembly of the chromatin fiber and higher-order chromatin structures. Thus, in order to provide a foundation for understanding these functions, we present a review of the basic elements of nucleosome structure and stability, including the association of linker histones. PMID:25980611
Prescott, Eugenia T; Safi, Alexias; Rusche, Laura N
Extended heterochromatin domains, which are repressive to transcription and help define centromeres and telomeres, are formed through specific interactions between silencing proteins and nucleosomes. This study reveals that in Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of multiple types of heterochromatin, but not for local repression mediated by a related transcriptional repressor. Thus, this region of the nucleosome may be generally important to long-range silencing. In S. cerevisiae, the Sir proteins perform long-range silencing, whereas the Sum1 complex acts locally to repress specific genes. A mutant form of Sum1p, Sum1-1p, achieves silencing in the absence of Sir proteins. A genetic screen identified mutations in histones H3 and H4 that disrupt Sum1-1 silencing and fall in regions of the nucleosome previously known to disrupt Sir silencing and rDNA silencing. In contrast, no mutations were identified that disrupt wild-type Sum1 repression. Mutations that disrupt silencing fall in two regions of the nucleosome, the tip of the H3 tail and a surface of the nucleosomal core (LRS domain) and the adjacent base of the H4 tail. The LRS/H4 tail region interacts with the Sir3p bromo-adjacent homology (BAH) domain to facilitate Sir silencing. By analogy, this study is consistent with the LRS/H4 tail region interacting with Orc1p, a paralog of Sir3p, to facilitate Sum1-1 silencing. Thus, the LRS/H4 tail region of the nucleosome may be relatively accessible and facilitate interactions between silencing proteins and nucleosomes to stabilize long-range silencing.
Tachiwana, Hiroaki; Kagawa, Wataru; Shiga, Tatsuya; Osakabe, Akihisa; Miya, Yuta; Saito, Kengo; Hayashi-Takanaka, Yoko; Oda, Takashi; Sato, Mamoru; Park, Sam-Yong; Kimura, Hiroshi; Kurumizaka, Hitoshi
In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.
Mavrich, Travis N; Ioshikhes, Ilya P; Venters, Bryan J; Jiang, Cizhong; Tomsho, Lynn P; Qi, Ji; Schuster, Stephan C; Albert, Istvan; Pugh, B Franklin
Most nucleosomes are well-organized at the 5' ends of S. cerevisiae genes where "-1" and "+1" nucleosomes bracket a nucleosome-free promoter region (NFR). How nucleosomal organization is specified by the genome is less clear. Here we establish and inter-relate rules governing genomic nucleosome organization by sequencing DNA from more than one million immunopurified S. cerevisiae nucleosomes (displayed at http://atlas.bx.psu.edu/). Evidence is presented that the organization of nucleosomes throughout genes is largely a consequence of statistical packing principles. The genomic sequence specifies the location of the -1 and +1 nucleosomes. The +1 nucleosome forms a barrier against which nucleosomes are packed, resulting in uniform positioning, which decays at farther distances from the barrier. We present evidence for a novel 3' NFR that is present at >95% of all genes. 3' NFRs may be important for transcription termination and anti-sense initiation. We present a high-resolution genome-wide map of TFIIB locations that implicates 3' NFRs in gene looping.
Öztürk, Mehmet Ali; Pachov, Georgi V; Wade, Rebecca C; Cojocaru, Vlad
Linker histones are essential for DNA compaction in chromatin. They bind to nucleosomes in a 1:1 ratio forming chromatosomes. Alternative configurations have been proposed in which the globular domain of the linker histone H5 (gH5) is positioned either on- or off-dyad between the nucleosomal and linker DNAs. However, the dynamic pathways of chromatosome assembly remain elusive. Here, we studied the conformational plasticity of gH5 in unbound and off-dyad nucleosome-bound forms with classical and accelerated molecular dynamics simulations. We find that the unbound gH5 converts between open and closed conformations, preferring the closed form. However, the open gH5 contributes to a more rigid chromatosome and restricts the motion of the nearby linker DNA through hydrophobic interactions with thymidines. Moreover, the closed gH5 opens and reorients in accelerated simulations of the chromatosome. Brownian dynamics simulations of chromatosome assembly, accounting for a range of amplitudes of nucleosome opening and different nucleosome DNA sequences, support the existence of both on- and off-dyad binding modes of gH5 and reveal alternative, sequence and conformation-dependent chromatosome configurations. Taken together, these findings suggest that the conformational dynamics of linker histones and nucleosomes facilitate alternative chromatosome configurations through an interplay between induced fit and conformational selection. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Yue, Hongjun; Fang, He; Wei, Sijie; Hayes, Jeffrey J; Lee, Tae-Hee
Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.
Yang, Xi; Yan, Yan
The histone octamer induced bending of DNA into the super-helix structure in nucleosome core particle, is very unique and vital for DNA packing into chromatin. We collected 48 nucleosome crystal structures from PDB and applied a multivariate analysis on the nucleosome structural data. Based on the anisotropic nature of DNA structure, a principal conformational subspace (PCS) is derived from multiple properties to represent the most significant variances of nucleosome DNA structures. The coupling of base pair-oriented parameters with sugar phosphate backbone parameters presented in principal dimensionalities reveals two main deformation modes that have supplemented the existing physical model. By using sequence alignment-based statistics, a positiondependent conformational map for the super-helical DNA path is established. The result shows that the crystal structures of nucleosome DNA have much consistency in position-specific structural variations and certain periodicity is found to exist in these variations. Thus, the positions with obvious deformation patterns along the DNA path in nucleosome core particle are relatively conservative from the perspective of statistics. PMID:22125381
Öztürk, Mehmet Ali; Pachov, Georgi V.; Wade, Rebecca C.; Cojocaru, Vlad
Linker histones are essential for DNA compaction in chromatin. They bind to nucleosomes in a 1:1 ratio forming chromatosomes. Alternative configurations have been proposed in which the globular domain of the linker histone H5 (gH5) is positioned either on- or off-dyad between the nucleosomal and linker DNAs. However, the dynamic pathways of chromatosome assembly remain elusive. Here, we studied the conformational plasticity of gH5 in unbound and off-dyad nucleosome-bound forms with classical and accelerated molecular dynamics simulations. We find that the unbound gH5 converts between open and closed conformations, preferring the closed form. However, the open gH5 contributes to a more rigid chromatosome and restricts the motion of the nearby linker DNA through hydrophobic interactions with thymidines. Moreover, the closed gH5 opens and reorients in accelerated simulations of the chromatosome. Brownian dynamics simulations of chromatosome assembly, accounting for a range of amplitudes of nucleosome opening and different nucleosome DNA sequences, support the existence of both on- and off-dyad binding modes of gH5 and reveal alternative, sequence and conformation-dependent chromatosome configurations. Taken together, these findings suggest that the conformational dynamics of linker histones and nucleosomes facilitate alternative chromatosome configurations through an interplay between induced fit and conformational selection. PMID:27270081
Möbius, Wolfram; Osberg, Brendan; Tsankov, Alexander M.; Rando, Oliver J.; Gerland, Ulrich
Recent genome-wide maps of nucleosome positions in different eukaryotes have revealed a common pattern around transcription start sites, involving a nucleosome-free region flanked by a pronounced periodic pattern in the average nucleosome density. For the yeast S. cerevisiae, a gas of hard rods, known as Tonks gas and equivalent to the statistical positioning mechanism of Kornberg and Stryer, can be used to describe the experimentally observed pattern. Here, we consider 12 Hemiascomycota yeast species, each of which displays a distinct nucleosome pattern. Since the mechanisms underlying the formation of the patterns are expected to be related, we undertake a data-driven search for a unified quantitative description. We find that the simple one-dimensional gas model needs to be extended to take into account transient unwrapping of short segments of nucleosomal DNA, such that the particles no longer have a fixed size. Chromatin behavior in all but one species is well described by this generalized gas model, with a single unified set of model parameters where only the average nucleosome density is a species-dependent variable.
North, Justin A.; Shimko, John C.; Javaid, Sarah; Mooney, Alex M.; Shoffner, Matthew A.; Rose, Sean D.; Bundschuh, Ralf; Fishel, Richard; Ottesen, Jennifer J.; Poirier, Michael G.
Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2–hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry–exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair. PMID:22965129
Nissen, K.A.; Lan, S.Y.; Smerdon, M.J.
Rearrangements of chromatin structure during excision repair of UV-damaged DNA appear to involve unfolding of nucleosomal DNA while repair is taking place, followed by refolding of this DNA into a native nucleosome structure. Recently, we found that repair patches are not distributed uniformly along the DNA in nucleosome core particles immediately following their refolding into nucleosomes. Therefore, the distribution of repair patches in nucleosome core DNA was used to monitor the stability of nucleosome placement in these regions. Our results indicate that in nondividing human cells undergoing excision repair there is a slow change in the positioning of nucleosomes in newly repaired regions of chromatin, resulting in the eventual randomization of repair patches in nucleosome core DNA. Furthermore, the nonrandom placement of nucleosomes observed just after the refolding event is not re-established during DNA replication. Possible mechanisms for this change in nucleosome placement along the DNA are discussed.
Thakar, Amit; Parvin, Jeffrey D; Zlatanova, Jordanka
BRCA1, the protein product of the Breast Cancer Susceptibility Gene (BRCA1) has been implicated in multiple pathways that preserve genome stability, including cell cycle control, DNA repair, transcription, and chromatin remodeling. BRCA1, in complex with another RING-domain protein BARD1, possesses ubiquitin-ligase activity. Only a few targets for this activity have been identified in vivo. Nucleosomal histones may also be targets in vivo since they can be modified by the BRCA1/BARD1 complex in vitro. Here we demonstrate that the BRCA1/BARD1 complex can ubiquitylate both free H2A and H2B histones and histones in the context of nucleosomal particles. These results raise the possibility that BRCA1/BARD1 can directly affect nucleosomal structure, dynamics, and function through its ability to modify nucleosomal histones.
Riedmann, Caitlyn; Fondufe-Mittendorf, Yvonne N.
Chromatin architectural proteins (CAPs) bind the entry/exit DNA of nucleosomes and linker DNA to form higher order chromatin structures with distinct transcriptional outcomes. How CAPs mediate nucleosome dynamics is not well understood. We hypothesize that CAPs regulate DNA target site accessibility through alteration of the rate of spontaneous dissociation of DNA from nucleosomes. We investigated the effects of histone H1, high mobility group D1 (HMGD1), and methyl CpG binding protein 2 (MeCP2), on the biophysical properties of nucleosomes and chromatin. We show that MeCP2, like the repressive histone H1, traps the nucleosome in a more compact mononucleosome structure. Furthermore, histone H1 and MeCP2 hinder model transcription factor Gal4 from binding to its cognate DNA site within the nucleosomal DNA. These results demonstrate that MeCP2 behaves like a repressor even in the absence of methylation. Additionally, MeCP2 behaves similarly to histone H1 and HMGD1 in creating a higher-order chromatin structure, which is susceptible to chromatin remodeling by ISWI. Overall, we show that CAP binding results in unique changes to nucleosome structure and dynamics. PMID:27624769
Zouein, Fouad A.; Zgheib, Carlos; Hamza, Shereen; Fuseler, John W.; Hall, John E.; Soljancic, Andrea; Lopez-Ruiz, Arnaldo; Kurdi, Mazen; Booz, George W.
STAT3 is involved in protection of the heart provided by ischemic preconditioning. However, the role of this transcription factor in the heart in chronic stresses such as hypertension has not been defined. We assessed whether STAT3 is important in hypertension-induced cardiac remodeling using mice with reduced STAT3 activity due to a S727A mutation (SA/SA). Wild type (WT) and SA/SA mice received angiotensin (ANG) II or saline for 17 days. ANG II increased mean arterial and systolic pressure in SA/SA and WT mice, but cardiac levels of cytokines associated with heart failure were increased less in SA/SA mice. Unlike WT mice, hearts of SA/SA mice showed signs of developing systolic dysfunction as evidenced by reduction in ejection fraction and fractional shortening. In the left ventricle of both WT and SA/SA mice, ANG II induced fibrosis. However, fibrosis in SA/SA mice appeared more extensive and was associated with loss of myocytes. Cardiac hypertrophy as indexed by heart to body weight ratio and left ventricular anterior wall dimension during diastole was greater in WT mice. In WT+ANG II mice there was an increase in the mass of individual myofibrils. In contrast, cardiac myocytes of SA/SA+ANG II mice showed a loss in myofibrils and myofibrillar mass density was decreased during ANG II infusion. Our findings reveal that STAT3 transcriptional activity is important for normal cardiac myocyte myofibril morphology. Loss of STAT3 may impair cardiac function in the hypertensive heart due to defective myofibrillar structure and remodeling that may lead to heart failure. PMID:23364341
Abu-Abied, Mohamad; Szwerdszarf, David; Mordehaev, Inna; Yaniv, Yossi; Levinkron, Saar; Rubinstein, Mor; Riov, Joseph; Ophir, Ron; Sadot, Einat
The ability to form adventitious roots (AR) is an economically important trait that is lost during the juvenile-to-mature phase change in woody plants. Auxin treatment, which generally promotes rooting in juvenile cuttings, is often ineffective when applied to mature cuttings. The molecular basis for this phenomenon in Eucalyptus grandis was addressed here. A comprehensive microarray analysis was performed in order to compare gene-expression profiles in juvenile and mature cuttings of E. grandis, with or without auxin treatment on days, 0, 1, 3, 6, 9 and 12 post AR induction. Under these conditions AR primordia were formed only in auxin-treated juvenile cuttings. However, clustering the expression profiles revealed that the time after induction contributed more significantly to the differences in expression than the developmental phase of the cuttings or auxin treatment. Most detected differences which were related to the developmental phase and auxin treatment occurred on day 6, which correlated with the kinetics of AR-primordia formation. Among the functional groups of transcripts that differed between juvenile and mature cuttings was that of microtubules (MT). The expression of 42 transcripts annotated as coding for tubulin, MT-associated proteins and kinesin motor proteins was validated in the same RNA samples. The results suggest a coordinated developmental and auxin dependent regulation of several MT-related transcripts in these cuttings. To determine the relevance of MT remodeling to AR formation, MTs were subjected to subtle perturbations by trifluralin, a MT disrupting drug, applied during auxin induction. Juvenile cuttings were not affected by the treatment, but rooting of mature cuttings increased from 10 to more than 40 percent. The data suggest that juvenile-specific MT remodeling is involved in AR formation in E. grandis.
Galati, Alessandra; Micheli, Emanuela; Alicata, Claudia; Ingegnere, Tiziano; Cicconi, Alessandro; Pusch, Miriam Caroline; Giraud-Panis, Marie-Josèphe; Gilson, Eric; Cacchione, Stefano
The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Galati, Alessandra; Micheli, Emanuela; Alicata, Claudia; Ingegnere, Tiziano; Cicconi, Alessandro; Pusch, Miriam Caroline; Giraud-Panis, Marie-Josèphe; Gilson, Eric; Cacchione, Stefano
The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection. PMID:25999344
Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I.; Patel, Smita S.; Studitsky, Vasily M.
Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31–40) and +(41–65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop. PMID:27847876
Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin
A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.
Bao, Yunhe; Shen, Xuetong
ATP-dependent chromatin remodeling complexes use ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. However, emerging lines of evidence suggest that chromatin remodeling complexes are important players in DNA double-strand break (DSB) repair as well. The INO80 and SWI2 subfamilies of chromatin remodeling complexes have been found to be recruited to the double-strand lesions and to function directly in both homologous recombination and non-homologous end-joining, the two major conserved DSB repair pathways. Improperly repaired DSBs are implicated in cancer development in higher organisms. Understanding how chromatin remodeling complexes contribute to DSB repair should provide new insights into the mechanisms of carcinogenesis and might suggest new targets for cancer treatment.
Rogge, Ryan A; Kalashnikova, Anna A; Muthurajan, Uma M; Porter-Goff, Mary E; Luger, Karolin; Hansen, Jeffrey C
Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.
Gentry, Matthew; Hennig, Lars
Establishment and dynamic regulation of a higher order chromatin structure is an essential component of development. Chromatin remodelling complexes such as the SWI2/SNF2 family of ATP-dependent chromatin remodellers can alter chromatin architecture by changing nucleosome positioning or substituting histones with histone variants. These remodellers often act in concert with chromatin modifiers such as the polycomb group proteins which confer repressive states through modification of histone tails. These mechanisms are highly conserved across the eukaryotic kingdom although in plants, owing to the maintenance of dedifferentiated cell states that allow for post-embyronic changes in development, strict control of chromatin remodelling is even more paramount. Recent and ongoing studies in the model plant Arabidopsis thaliana have found that while the major families of the SWI2/SNF2 ATPase chromatin remodellers are represented, a number of redundancies and divergent functions have emerged that show a break from the roles of their metazoan counterparts. This review focusses on the SNF2 and CHD families of ATP-dependent remodellers and their roles in plant development. © 2013 Published by Elsevier Inc.
All the DNA in a cell's nucleus is packaged into a material called chromatin consisting of DNA and DNA-associated proteins. The basic unit of chromatin is the nucleosome which consists of 147 bases of DNA wrapped around a protein core composed of two copies each of histones H2A, H2B, H3, and H4. DNA wrapped into a nucleosome is inaccessible to most DNA processing machinery. This machinery needs access to the DNA to perform processes such as transcription, replication, and repair. The cell uses many mechanisms to modulate this protection including nucleosome unwrapping, sliding, remodeling, disassembly, and chemical modification. This work used transcription factors along with fluorescent methods to probe the accessibility of DNA near the ends of the nucleosome. The DNA in this entry-exit region can spontaneously unwrap to provide transient access to DNA binding factors such as transcription factors. We studied the effect of histone post-translational modifications on the accessibility of DNA in the entry-exit region. We found that the phosphorylation at H3Y41ph increased DNA accessibility in vitro by 3-fold, which is similar in size to the effect of a previously studied modification H3K56ac. Since both of these modifications are associated with active genes and could occur together, we studied nucleosomes with both modifications and they showed a 17+/-5-fold increase in accessibility. This indicates that the cell could use multiple modifications in the entry-exit region to adjust the accessibility of nucleosomal DNA by over an order of magnitude. We also studied the effects of the removal of one H2A/H2B dimer. These partially formed nucleosomes, called hexasomes, are a component of chromatin that has been largely unstudied. Our results have shown that hexasomes have DNA accessibility similar to that of fully-formed nucleosomes on the side with the remaining H2A/H2B dimer. However, on the side without the dimer, our results showed the DNA remains permanently
The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication.
The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication.
Clatterbuck Soper, Sarah F; Dator, Romel P; Limbach, Patrick A; Woodson, Sarah A
Assembly of 30S ribosomal subunits from their protein and RNA components requires extensive refolding of the 16S rRNA and is assisted by 10-20 assembly factors in bacteria. We probed the structures of 30S assembly intermediates in E. coli cells, using a synchrotron X-ray beam to generate hydroxyl radical in the cytoplasm. Widespread differences between mature and pre-30S complexes in the absence of assembly factors RbfA and RimM revealed global reorganization of RNA-protein interactions prior to maturation of the 16S rRNA and showed how RimM reduces misfolding of the 16S 3' domain during transcription in vivo. Quantitative (14)N/(15)N mass spectrometry of affinity-purified pre-30S complexes confirmed the absence of tertiary assembly proteins and showed that N-terminal acetylation of proteins S18 and S5 correlates with correct folding of the platform and central pseudoknot. Our results indicate that cellular factors delay specific RNA folding steps to ensure the quality of assembly. Copyright © 2013 Elsevier Inc. All rights reserved.
Trifonov, Edward N
Thorough quantitative study of nucleosome repeat length (NRL) distributions, conducted in 1992 by J. Widom, resulted in a striking observation that the linker lengths between the nucleosomes are quantized. Comparison of the NRL average values with the MNase cut distances predicted from the hypothetical columnar structure of chromatin (this work) shows a close correspondence between the two. This strongly suggests that the NRL distribution, actually, reflects the dominant role of columnar chromatin structure common for all eukaryotes.
Klinker, Henrike; Mueller-Planitz, Felix; Yang, Renliang; Forné, Ignasi; Liu, Chuan-Fa; Nordenskiöld, Lars; Becker, Peter B.
ISWI is the catalytic subunit of several ATP-dependent chromatin remodelling factors that catalyse the sliding of nucleosomes along DNA and thereby endow chromatin with structural flexibility. Full activity of ISWI requires residues of a basic patch of amino acids in the N-terminal ‘tail’ of histone H4. Previous studies employing oligopeptides and mononucleosomes suggested that acetylation of the H4 tail at lysine 16 (H4K16) within the basic patch may inhibit the activity of ISWI. On the other hand, the acetylation of H4K16 is known to decompact chromatin fibres. Conceivably, decompaction may enhance the accessibility of nucleosomal DNA and the H4 tail for ISWI interactions. Such an effect can only be evaluated at the level of nucleosome arrays. We probed the influence of H4K16 acetylation on the ATPase and nucleosome sliding activity of Drosophila ISWI in the context of defined, in vitro reconstituted chromatin fibres with physiological nucleosome spacing and linker histone content. Contrary to widespread expectations, the acetylation did not inhibit ISWI activity, but rather stimulated ISWI remodelling under certain conditions. Therefore, the effect of H4K16 acetylation on ISWI remodelling depends on the precise nature of the substrate. PMID:24516652
Brysbaert, Guillaume; Lensink, Marc F.; Blossey, Ralf
Recently, kinetic proofreading scenarios have been proposed for the regulation of chromatin remodeling, first on purely theoretical grounds (Blossey and Schiessel 2008 HFSP J. 2 167-70) and deduced from experiments on the ISWI/ACF system (Narlikar 2010 Curr. Opin. Chem. Biol. 14 660). In the kinetic proofreading scenario of chromatin remodeling, the combination of the recognition of a histone tail state and ATP-hydrolysis in the remodeler motor act together to select (i.e. proofread) a nucleosomal substrate. ISWI remodelers have recently been shown to have an additional level of regulation as they contain auto-inhibitory motifs which need to be inactivated through an interaction with the nucleosome. In this paper we show that the auto-regulatory effect enhances substrate recognition in kinetic proofreading. We further report some suggestive additional insights into the molecular mechanism underlying ISWI-autoregulation.
Gursoy-Yuzugullu, Ozge; House, Nealia; Price, Brendan D
The ability of cells to detect and repair DNA double-strand breaks (DSBs) is dependent on reorganization of the surrounding chromatin structure by chromatin remodeling complexes. These complexes promote access to the site of DNA damage, facilitate processing of the damaged DNA and, importantly, are essential to repackage the repaired DNA. Here, we will review the chromatin remodeling steps that occur immediately after DSB production and that prepare the damaged chromatin template for processing by the DSB repair machinery. DSBs promote rapid accumulation of repressive complexes, including HP1, the NuRD complex, H2A.Z and histone methyltransferases at the DSB. This shift to a repressive chromatin organization may be important to inhibit local transcription and limit mobility of the break and to maintain the DNA ends in close contact. Subsequently, the repressive chromatin is rapidly dismantled through a mechanism involving dynamic exchange of the histone variant H2A.Z. H2A.Z removal at DSBs alters the acidic patch on the nucleosome surface, promoting acetylation of the H4 tail (by the NuA4-Tip60 complex) and shifting the chromatin to a more open structure. Further, H2A.Z removal promotes chromatin ubiquitination and recruitment of additional DSB repair proteins to the break. Modulation of the nucleosome surface and nucleosome function during DSB repair therefore plays a vital role in processing of DNA breaks. Further, the nucleosome surface may function as a central hub during DSB repair, directing specific patterns of histone modification, recruiting DNA repair proteins and modulating chromatin packing during processing of the damaged DNA template.
Fournier, Joëlle; Teillet, Alice; Chabaud, Mireille; Ivanov, Sergey; Genre, Andrea; Limpens, Erik; de Carvalho-Niebel, Fernanda; Barker, David G
In many legumes, root entry of symbiotic nitrogen-fixing rhizobia occurs via host-constructed tubular tip-growing structures known as infection threads (ITs). Here, we have used a confocal microscopy live-tissue imaging approach to investigate early stages of IT formation in Medicago truncatula root hairs (RHs) expressing fluorescent protein fusion reporters. This has revealed that ITs only initiate 10 to 20 h after the completion of RH curling, by which time major modifications have occurred within the so-called infection chamber, the site of bacterial entrapment. These include the accumulation of exocytosis (M. truncatula Vesicle-Associated Membrane Protein721e)- and cell wall (M. truncatula EARLY NODULIN11)-associated markers, concomitant with radial expansion of the chamber. Significantly, the infection-defective M. truncatula nodule inception-1 mutant is unable to create a functional infection chamber. This underlines the importance of the NIN-dependent phase of host cell wall remodeling that accompanies bacterial proliferation and precedes IT formation, and leads us to propose a two-step model for rhizobial infection initiation in legume RHs.
Zhou, Bing-Rui; Feng, Hanqiao; Ghirlando, Rodolfo; Kato, Hidenori; Gruschus, James; Bai, Yawen
Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K(16)R(17)H(18)R(19) in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K(16)R(17)H(18)R(19). Published by Elsevier Ltd.
Easley, Rebecca; Carpio, Lawrence; Dannenberg, Luke; Choi, Soyun; Alani, Dowser; Van Duyne, Rachel; Guendel, Irene; Klase, Zachary; Agbottah, Emmanuel; Kehn-Hall, Kylene; Kashanchi, Fatah
The SWI/SNF complex remodels nucleosomes, allowing RNA Polymerase II access to the HIV-1 proviral DNA. It has not been determined which SWI/SNF complex (BAF or PBAF) remodels nucleosomes at the transcription start site. These complexes differ in only three subunits and determining which subunit(s) is required could explain the regulation of Tat activated transcription. We show that PBAF is required for chromatin remodeling at the nuc-1 start site and transcriptional elongation. We find that Baf200 is required to ensure activation at the LTR level and for viral production. Interestingly, the BAF complex was observed on the LTR whereas PBAF was present on both LTR and Env regions. We found that Tat activated transcription facilitates removal of histones H2A and H2B at the LTR, and that the FACT complex may be responsible for their removal. Finally, the BAF complex may play an important role in regulating splicing of the HIV-1 genome. PMID:20599239
Knezetic, J.A.; Jacob, G.A.; Luse, D.S.
The authors have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, they demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that the observations are not the result of slow displacemnt of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.
Zhou, Coral Y; Johnson, Stephanie L; Gamarra, Nathan I; Narlikar, Geeta J
Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.
McGinty, Robert K.; Henrici, Ryan C.; Tan, Song
The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in higher eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the Ring1B/Bmi1/UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with multiple nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome. PMID:25355358
Vlijm, Rifka; Lee, Mina; Lipfert, Jan; Lusser, Alexandra; Dekker, Cees; Dekker, Nynke H
DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length of single DNA molecules, we monitor the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1). Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state (ΔLk = -0.73) and a right-handed state (ΔLk = +1.0), separated by a free energy difference of 2.3 kBT (1.5 kcal/mol). This flipping occurs without concomitant changes in DNA end-to-end length. The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. Our findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a "twist reservoir," offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.
McGinty, Robert K; Henrici, Ryan C; Tan, Song
The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in many eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys 119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the human Ring1B-Bmi1-UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with several nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome.
Li, Qing; Zhou, Hui; Wurtele, Hugo; Davies, Brian; Horazdovsky, Bruce; Verreault, Alain; Zhang, Zhiguo
SUMMARY Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106. PMID:18662540
Bowerman, Samuel; Wereszczynski, Jeff
Eukaryotes tune the transcriptional activity of their genome by altering the nucleosome core particle through multiple chemical processes. In particular, replacement of the canonical H2A histone with the variants macroH2A and H2A.Z has been shown to affect DNA accessibility and nucleosome stability; however, the processes by which this occurs remain poorly understood. Here, we elucidate the molecular mechanisms of these variants with an extensive molecular dynamics study of the canonical nucleosome along with three variant-containing structures: H2A.Z, macroH2A, and an H2A mutant with macroH2A-like L1 loops. Simulation results show that variant L1 loops play a pivotal role in stabilizing DNA binding to the octamer through direct interactions, core structural rearrangements, and altered allosteric networks in the nucleosome. All variants influence dynamics; however, macroH2A-like systems have the largest effect on energetics. In addition, we provide a comprehensive analysis of allosteric networks in the nucleosome and demonstrate that variants take advantage of stronger interactions between L1 loops to propagate dynamics throughout the complex. Furthermore, we show that post-translational modifications are enriched at key locations in these networks. Taken together, these results provide new insights into the relationship between the structure, dynamics, and function of the nucleosome core particle and chromatin fibers, and how they are influenced by chromatin remodelling factors.
Bowerman, Samuel; Wereszczynski, Jeff
Eukaryotes tune the transcriptional activity of their genome by altering the nucleosome core particle through multiple chemical processes. In particular, replacement of the canonical H2A histone with the variants macroH2A and H2A.Z has been shown to affect DNA accessibility and nucleosome stability; however, the processes by which this occurs remain poorly understood. In this study, we elucidate the molecular mechanisms of these variants with an extensive molecular dynamics study of the canonical nucleosome along with three variant-containing structures: H2A.Z, macroH2A, and an H2A mutant with macroH2A-like L1 loops. Simulation results show that variant L1 loops play a pivotal role in stabilizing DNA binding to the octamer through direct interactions, core structural rearrangements, and altered allosteric networks in the nucleosome. All variants influence dynamics; however, macroH2A-like systems have the largest effect on energetics. In addition, we provide a comprehensive analysis of allosteric networks in the nucleosome and demonstrate that variants take advantage of stronger interactions between L1 loops to propagate dynamics throughout the complex. Furthermore, we show that posttranslational modifications are enriched at key locations in these networks. Taken together, these results provide, to our knowledge, new insights into the relationship between the structure, dynamics, and function of the nucleosome core particle and chromatin fibers, and how they are influenced by chromatin remodeling factors.
Han, Soon-Ki; Wu, Miin-Feng; Cui, Sujuan; Wagner, Doris
Chromatin remodeling ATPases and their associated complexes can alter the accessibility of the genome in the context of chromatin by using energy derived from the hydrolysis of ATP to change the positioning, occupancy and composition of nucleosomes. In animals and plants, these remodelers have been implicated in diverse processes ranging from stem cell maintenance and differentiation to developmental phase transitions and stress responses. Detailed investigation of their roles in individual processes has suggested a higher level of selectivity of chromatin remodeling ATPase activity than previously anticipated, and diverse mechanisms have been uncovered that can contribute to the selectivity. This review summarizes recent advances in understanding the roles and activities of chromatin remodeling ATPases in plants.
Bazett-Jones, D P; Mendez, E; Czarnota, G J; Ottensmeyer, F P; Allfrey, V G
We have characterized the structure of transcriptionally active nucleosome subunits using electron spectroscopic imaging. Individual nucleosomes were analyzed in terms of total mass, DNA and protein content, while the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleosomes were separated from inactive nucleosomes by mercury-affinity chromatography thus making it possible to compare their structures. The chromatographic results combined with electron spectroscopic imaging confirm that active nucleosomes unfold to form extended U-shaped particles. Phosphorus mapping indicated that the nucleosomal DNA also underwent a conformational change consistent with particle unfolding. The three-dimensional structure of the Hg-affinity purified nucleosomes determined using quaternion-assisted angular reconstitution methods unites and resolves the different electron microscopic views of the particle and is concordant with a sulphydryl-exposing disruption of the H3-H4 tetramer. PMID:8628657
Suzuki, Yuya; Horikoshi, Naoki; Kato, Daiki; Kurumizaka, Hitoshi
The crotonylation of histones is an important post-translational modification, and epigenetically functions in the regulation of genomic DNA activity. The histone modifications in the structured "histone-fold" domains are considered to have an especially important impact on the nucleosome structure and dynamics. In the present study, we reconstituted the human nucleosome containing histone H3.2 crotonylated at the Lys122 residue, and determined its crystal structure at 2.56 Å resolution. We found that the crotonylation of the H3 Lys122 residue does not affect the overall nucleosome structure, but locally impedes the formation of the water-mediated hydrogen bond with the DNA backbone. Consistently, thermal stability assays revealed that the H3 Lys122 crotonylation, as well as the H3 Lys122 acetylation, clearly reduced the histone-DNA association. Copyright © 2015 Elsevier Inc. All rights reserved.
Kato, Hidenori; Jiang, Jiansheng; Zhou, Bing-Rui; Rozendaal, Marieke; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T. Sam; Straight, Aaron F.; Bai, Yawen
Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Key to kinetochore assembly is the specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A/H2B. We further find that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through nucleosome-docking-facilitated hydrophobic interactions. PMID:23723239
Zocco, Manuel; Marasovic, Mirela; Pisacane, Paola; Bilokapic, Silvija; Halic, Mario
To maintain genome stability, cells pack large portions of their genome into silent chromatin or heterochromatin. Histone H3 lysine 9 methylation, a hallmark of heterochromatin, is recognized by conserved readers called chromodomains. But how chromodomains interact with their actual binding partner, the H3K9 methylated nucleosome, remains elusive. We have determined the structure of a nucleosome trimethylated at lysine 9 of histone H3 (H3K9me3 Nucleosome) in a complex with the chromodomain of Chp1, a protein required for RNA interference-dependent heterochromatin formation in fission yeast. The cryo-electron microscopy structure reveals that the chromodomain of Chp1 binds the histone H3 lysine 9 methylated tail and the core of the nucleosome, primarily histones H3 and H2B. Mutations in chromodomain of Chp1 loops, which interact with the nucleosome core, abolished this interaction in vitro. Moreover, fission yeast cells with Chp1 loop mutations have a defect in Chp1 recruitment and heterochromatin formation. This study reveals the structural basis for heterochromatic silencing and suggests that chromodomains could read histone code in the H3 tail and the nucleosome core, which would provide an additional layer of regulation. PMID:27462451
Reynolds, Sheila M.; Weng, Zhiping; Bilmes, Jeff A.; Noble, William Stafford
We describe a probabilistic model, implemented as a dynamic Bayesian network, that can be used to predict nucleosome positioning along a chromosome based on one or more genomic input tracks containing position-specific information (evidence). Previous models have either made predictions based on primary DNA sequence alone, or have been used to infer nucleosome positions from experimental data. Our framework permits the combination of these two distinct types of information. We show how this flexible framework can be used to make predictions based on either sequence-model scores or experimental data alone, or by using the two in combination to interpret the experimental data and fill in gaps. The model output represents the posterior probability, at each position along the chromosome, that a nucleosome core overlaps that position, given the evidence. This posterior probability is computed by integrating the information contained in the input evidence tracks along the entire input sequence, and fitting the evidence to a simple grammar of alternating nucleosome cores and linkers. In addition to providing a novel mechanism for the prediction of nucleosome positioning from arbitrary heterogeneous data sources, this framework is also applicable to other genomic segmentation tasks in which local scores are available from models or from data that can be interpreted as defining a probability assignment over labels at that position. The ability to combine sequence-based predictions and data from experimental assays is a significant and novel contribution to the ongoing research regarding the primary structure of chromatin and its effects upon gene regulation.
Cutter, Amber R; Hayes, Jeffrey J
The nucleosomal subunit organization of chromatin provides a multitude of functions. Nucleosomes elicit an initial ∼7-fold linear compaction of genomic DNA. They provide a critical mechanism for stable repression of genes and other DNA-dependent activities by restricting binding of trans-acting factors to cognate DNA sequences. Conversely they are engineered to be nearly meta-stable and disassembled (and reassembled) in a facile manner to allow rapid access to the underlying DNA during processes such as transcription, replication and DNA repair. Nucleosomes protect the genome from DNA damaging agents and provide a lattice onto which a myriad of epigenetic signals are deposited. Moreover, vast strings of nucleosomes provide a framework for assembly of the chromatin fiber and higher-order chromatin structures. Thus, in order to provide a foundation for understanding these functions, we present a review of the basic elements of nucleosome structure and stability, including the association of linker histones. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Collino, Sebastiano; Montoliu, Ivan; Martin, François-Pierre J.; Scherer, Max; Mari, Daniela; Salvioli, Stefano; Bucci, Laura; Ostan, Rita; Monti, Daniela; Biagi, Elena; Brigidi, Patrizia; Franceschi, Claudio; Rezzi, Serge
The aging phenotype in humans has been thoroughly studied but a detailed metabolic profiling capable of shading light on the underpinning biological processes of longevity is still missing. Here using a combined metabonomics approach compromising holistic 1H-NMR profiling and targeted MS approaches, we report for the first time the metabolic phenotype of longevity in a well characterized human aging cohort compromising mostly female centenarians, elderly, and young individuals. With increasing age, targeted MS profiling of blood serum displayed a marked decrease in tryptophan concentration, while an unique alteration of specific glycerophospholipids and sphingolipids are seen in the longevity phenotype. We hypothesized that the overall lipidome changes specific to longevity putatively reflect centenarians' unique capacity to adapt/respond to the accumulating oxidative and chronic inflammatory conditions characteristic of their extreme aging phenotype. Our data in centenarians support promotion of cellular detoxification mechanisms through specific modulation of the arachidonic acid metabolic cascade as we underpinned increased concentration of 8,9-EpETrE, suggesting enhanced cytochrome P450 (CYP) enzyme activity. Such effective mechanism might result in the activation of an anti-oxidative response, as displayed by decreased circulating levels of 9-HODE and 9-oxoODE, markers of lipid peroxidation and oxidative products of linoleic acid. Lastly, we also revealed that the longevity process deeply affects the structure and composition of the human gut microbiota as shown by the increased extrection of phenylacetylglutamine (PAG) and p-cresol sulfate (PCS) in urine of centenarians. Together, our novel approach in this representative Italian longevity cohort support the hypothesis that a complex remodeling of lipid, amino acid metabolism, and of gut microbiota functionality are key regulatory processes marking exceptional longevity in humans. PMID:23483888
Grene, Ruth; Klumas, Curtis; Suren, Haktan; Yang, Kuan; Collakova, Eva; Myers, Elijah; Heath, Lenwood S.; Holliday, Jason A.
Microarray gene expression profiling is a powerful technique to understand complex developmental processes, but making biologically meaningful inferences from such studies has always been challenging. We previously reported a microarray study of the freezing acclimation period in Sitka spruce (Picea sitchensis) in which a large number of candidate genes for climatic adaptation were identified. In the current paper, we apply additional systems biology tools to these data to further probe changes in the levels of genes and metabolites and activities of associated pathways that regulate this complex developmental transition. One aspect of this adaptive process that is not well understood is the role of the cell wall. Our data suggest coordinated metabolic and signaling responses leading to cell wall remodeling. Co-expression of genes encoding proteins associated with biosynthesis of structural and non-structural cell wall carbohydrates was observed, which may be regulated by ethylene signaling components. At the same time, numerous genes, whose products are putatively localized to the endomembrane system and involved in both the synthesis and trafficking of cell wall carbohydrates, were up-regulated. Taken together, these results suggest a link between ethylene signaling and biosynthesis, and targeting of cell wall related gene products during the period of winter hardening. Automated Layout Pipeline for Inferred NEtworks (ALPINE), an in-house plugin for the Cytoscape visualization environment that utilizes the existing GeneMANIA and Mosaic plugins, together with the use of visualization tools, provided images of proposed signaling processes that became active over the time course of winter hardening, particularly at later time points in the process. The resulting visualizations have the potential to reveal novel, hypothesis-generating, gene association patterns in the context of targeted subcellular location. PMID:23112803
Rajashekar, Roopa; Liebl, David; Chikkaballi, Deepak; Liss, Viktoria; Hensel, Michael
Intracellular Salmonella enterica induce a massive remodeling of the endosomal system in infected host cells. One dramatic consequence of this interference is the induction of various extensive tubular aggregations of membrane vesicles, and tubules positive for late endosomal/lysosomal markers are referred to as Salmonella-induced filaments or SIF. SIF are highly dynamic in nature with extension and collapse velocities of 0.4-0.5 µm x sec-1. The induction of SIF depends on the function of the Salmonella Pathogenicity Island 2 (SPI2) encoded type III secretion system (T3SS) and a subset of effector proteins. In this study, we applied live cell imaging and electron microscopy to analyze the role of individual effector proteins in SIF morphology and dynamic properties of SIF. SIF in cells infected with sifB, sseJ, sseK1, sseK2, sseI, sseL, sspH1, sspH2, slrP, steC, gogB or pipB mutant strains showed a morphology and dynamics comparable to SIF induced by WT Salmonella. SIF were absent in cells infected with the sifA-deficient strain and live cell analyses allowed tracking of the loss of the SCV membrane of intracellular sifA Salmonella. In contrast to analyses in fixed cells, in living host cells SIF induced by sseF- or sseG-deficient strains were not discontinuous, but rather continuous and thinner in diameter. A very dramatic phenotype was observed for the pipB2-deficient strain that induced very bulky, non-dynamic aggregations of membrane vesicles. Our study underlines the requirement of the study of Salmonella-host interaction in living systems and reveals new phenotypes due to the intracellular activities of Salmonella.
Collino, Sebastiano; Montoliu, Ivan; Martin, François-Pierre J; Scherer, Max; Mari, Daniela; Salvioli, Stefano; Bucci, Laura; Ostan, Rita; Monti, Daniela; Biagi, Elena; Brigidi, Patrizia; Franceschi, Claudio; Rezzi, Serge
The aging phenotype in humans has been thoroughly studied but a detailed metabolic profiling capable of shading light on the underpinning biological processes of longevity is still missing. Here using a combined metabonomics approach compromising holistic (1)H-NMR profiling and targeted MS approaches, we report for the first time the metabolic phenotype of longevity in a well characterized human aging cohort compromising mostly female centenarians, elderly, and young individuals. With increasing age, targeted MS profiling of blood serum displayed a marked decrease in tryptophan concentration, while an unique alteration of specific glycerophospholipids and sphingolipids are seen in the longevity phenotype. We hypothesized that the overall lipidome changes specific to longevity putatively reflect centenarians' unique capacity to adapt/respond to the accumulating oxidative and chronic inflammatory conditions characteristic of their extreme aging phenotype. Our data in centenarians support promotion of cellular detoxification mechanisms through specific modulation of the arachidonic acid metabolic cascade as we underpinned increased concentration of 8,9-EpETrE, suggesting enhanced cytochrome P450 (CYP) enzyme activity. Such effective mechanism might result in the activation of an anti-oxidative response, as displayed by decreased circulating levels of 9-HODE and 9-oxoODE, markers of lipid peroxidation and oxidative products of linoleic acid. Lastly, we also revealed that the longevity process deeply affects the structure and composition of the human gut microbiota as shown by the increased extrection of phenylacetylglutamine (PAG) and p-cresol sulfate (PCS) in urine of centenarians. Together, our novel approach in this representative Italian longevity cohort support the hypothesis that a complex remodeling of lipid, amino acid metabolism, and of gut microbiota functionality are key regulatory processes marking exceptional longevity in humans.
Westin, L; Blomquist, P; Milligan, J F; Wrange, O
Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specific and required the oligonucleotide to be present during in vitro nucleosome reconstitution. Binding of the triple helix oligonucleotide on a 199 bp mouse mammary tumour virus promoter DNA fragment with a centrally located triplex DNA resulted in interruption of histone-DNA contacts flanking the triplex DNA segment. When nucleosome reconstitution is carried out on a longer, 279 bp DNA fragment with an asymmetrically located triplex site, nucleosome formation occurred at the border of the triple helical DNA. In this case the triplex DNA functioned as a nucleosome barrier. We conclude that triplex DNA cannot be accommodated within a nucleosome context and thus may be used to site-specifically manipulate nucleosome organization.
Westin, L; Blomquist, P; Milligan, J F; Wrange, O
Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specific and required the oligonucleotide to be present during in vitro nucleosome reconstitution. Binding of the triple helix oligonucleotide on a 199 bp mouse mammary tumour virus promoter DNA fragment with a centrally located triplex DNA resulted in interruption of histone-DNA contacts flanking the triplex DNA segment. When nucleosome reconstitution is carried out on a longer, 279 bp DNA fragment with an asymmetrically located triplex site, nucleosome formation occurred at the border of the triple helical DNA. In this case the triplex DNA functioned as a nucleosome barrier. We conclude that triplex DNA cannot be accommodated within a nucleosome context and thus may be used to site-specifically manipulate nucleosome organization. Images PMID:7610046
Jansen, An; van der Zande, Elisa; Meert, Wim; Fink, Gerald R; Verstrepen, Kevin J
The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called 'anti-nucleosomal elements' like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.
Ercan, Sevinc; Carrozza, Michael J; Workman, Jerry L
Recent studies show that active regulatory regions of the yeast genome have a lower density of nucleosomes than other regions, and that there is an inverse correlation between nucleosome density and the transcription rate of a gene. This may be the result of transcription factors displacing nucleosomes. PMID:15461807
Schwab, David; Bruinsma, Robijn
It has recently been shown that nucleosomes localize to preferred locations along DNA. This localization is a result of the sequence dependent bending stiffness of dsDNA, which must be wrapped around a histone protein to form a nucleosome. As a simple model of nucleosome localization, we study a one-dimensional hard-core gas in a random potential. We numerically solve for the density profile and other thermodynamic quantities using as input both randomly generated potential profiles and experimental energy landscapes. We compare with the annealed average, inspired by the Random Energy Model, and find that the quenched and annealed averages differ significantly above the localization temperature, implying sequence induced structural organization long before the system has frozen. Although information about the ground state is preserved at higher temperatures, there exist massive structural reorganizations at fixed temperature when the chemical potential is lowered. This offers another perspective on why different cells, with different chemical potentials, have different gene expression.
Hapala, Jan; Trifonov, Edward N
Transcription is known to be affected by the rotational setting of the transcription response elements within nucleosomes. We studied the rotational positioning of the TATA box, the most universal promoter motif. We applied a bioinformatic high-resolution nucleosome mapping technique to eukaryotic promoters. Our results show that the nucleosome DNA sequence harboring the TATA box encodes alternative rotational positions for the same piece of DNA. This may serve for switching the gene activity on and off. Copyright © 2013 Elsevier B.V. All rights reserved.
Benvenuto, Giovanna; Formiggini, Fabio; Laflamme, Pierre; Malakhov, Mikhail; Bowler, Chris
Light provides a major source of information from the environment during plant growth and development. Recent results suggest that the key events controlling light-regulated gene expression in plants are translocation of the phytochrome photoreceptors into the nucleus, followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and the COP9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness (e.g., ). Genetic analyses in Arabidopsis and tomato have revealed that the nuclear protein DET1 also plays a key role in the repression of photomorphogenesis. However, the function of this protein has remained a mystery. In a series of in vitro experiments, we provide persuasive evidence that DET1 binds to nonacetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, we have utilized FRET (fluorescence resonance energy transfer) imaging with GFP variants to demonstrate this interaction within the nucleus of living plant cells. Given the dramatic photomorphogenic phenotypes of det1 mutants, we propose that chromatin remodeling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis.
Warters, R.L.; Roti Roti, J.L.; Winward, R.T.
The effect of hyperthermia (40 to 80/sup 0/C) on the nucleosome structure of mammalian chromatin was determined using the enzyme micrococcal nuclease. At equivalent fractional DNA digestion it was found that neither the size of DNA nor the total fraction of cellular DNA associated with nucleosome structure is altered by heat exposure up to 48/sup 0/C for 30 min. It is proposed that this heat-induced reduction in the accessibility to nuclease attack of DNA in chromatin from heated cells is due to the increased protein mass associated with chromatin.
Corpet, Armelle; Almouzni, Geneviève
Understanding the basic mechanisms underlying chromatin dynamics during DNA replication in eukaryotic cells is of fundamental importance. Beyond DNA compaction, chromatin organization represents a means to regulate genome function. Thus, the inheritance and maintenance of the DNA sequence, along with its organization into chromatin, is central for eukaryotic life. To orchestrate DNA replication in the context of chromatin is a challenge, both in terms of accessibility to the compact structures and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome dynamics involving assembly pathways and chromatin maturation mechanisms that restore chromatin organization in the wake of DNA replication. In this review, we describe our current knowledge concerning how these pathways operate at the nucleosomal level and highlight the key players, such as histone chaperones, chromatin remodelers or modifiers, involved in the process of chromatin duplication. Major advances have been made recently concerning de novo nucleosome assembly and our understanding of its coordination with recycling of parental histones is progressing. Insights into the transmission of chromatin-based information during replication have important implications in the field of epigenetics to fully comprehend how the epigenetic landscape might, or at times might not, be stably maintained in the face of dramatic changes in chromatin structure.
Figueiredo, Luisa M; Cross, George A M
In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein VSG genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one VSG is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.
Yao, Wei; King, Devin A.; Beckwith, Sean L.; Gowans, Graeme J.; Yen, Kuangyu; Zhou, Coral
ATP-dependent chromatin remodeling complexes are essential for transcription regulation, and yet it is unclear how these multisubunit complexes coordinate their activities to facilitate diverse transcriptional responses. In this study, we found that the conserved Arp5 and Ies6 subunits of the Saccharomyces cerevisiae INO80 chromatin-remodeler form an abundant and distinct subcomplex in vivo and stimulate INO80-mediated activity in vitro. Moreover, our genomic studies reveal that the relative occupancy of Arp5-Ies6 correlates with nucleosome positioning at transcriptional start sites and expression levels of >1,000 INO80-regulated genes. Notably, these genes are significantly enriched in energy metabolism pathways. Specifically, arp5Δ, ies6Δ, and ino80Δ mutants demonstrate decreased expression of genes involved in glycolysis and increased expression of genes in the oxidative phosphorylation pathway. Deregulation of these metabolic pathways results in constitutively elevated mitochondrial potential and oxygen consumption. Our results illustrate the dynamic nature of the INO80 complex assembly and demonstrate for the first time that a chromatin remodeler regulates glycolytic and respiratory capacity, thereby maintaining metabolic stability. PMID:26755556
Kristell, Carolina; Orzechowski Westholm, Jakub; Olsson, Ida; Ronne, Hans; Komorowski, Jan; Bjerling, Pernilla
Gene transcription is associated with local changes in chromatin, both in nucleosome positions and in chemical modifications of the histones. Chromatin dynamics has mostly been studied on a single-gene basis. Those genome-wide studies that have been made primarily investigated steady-state transcription. However, three studies of genome-wide changes in chromatin during the transcriptional response to heat shock in the budding yeast Saccharomyces cerevisiae revealed nucleosome eviction in promoter regions but only minor effects in coding regions. Here, we describe the short-term response to nitrogen starvation in the fission yeast Schizosaccharomyces pombe. Nitrogen depletion leads to a fast induction of a large number of genes in S. pombe and is thus suitable for genome-wide studies of chromatin dynamics during gene regulation. After 20 min of nitrogen removal, 118 transcripts were up-regulated. The distribution of regulated genes throughout the genome was not random; many up-regulated genes were found in clusters, while large parts of the genome were devoid of up-regulated genes. Surprisingly, this up-regulation was associated with nucleosome eviction of equal magnitudes in the promoters and in the coding regions. The nucleosome loss was not limited to induction by nitrogen depletion but also occurred during cadmium treatment. Furthermore, the lower nucleosome density persisted for at least 60 min after induction. Two highly induced genes, urg1+ and urg2+, displayed a substantial nucleosome loss, with only 20% of the nucleosomes being left in the coding region. We conclude that nucleosome loss during transcriptional activation is not necessarily limited to promoter regions. PMID:20086243
Narlikar, Geeta J
ATP-dependent chromatin remodeling motors play fundamental roles in nuclear processes by regulating access to DNA. Yet compared to other cellular motors less is known about how these motors couple the energy of ATP to alter their substrates. Here we use recent studies on a key chromatin remodeling motor from the ISWI class, human ACF and its yeast counterpart, ISW2, to propose a model for how these motors use ATP to read structural cues presented by nucleosomal substrates. Substantial earlier work has shown that ACF activity is strongly regulated by the length of the DNA flanking a nucleosome as well as by the histone H4 tail. Recent bulk and single-molecule studies of human ACF suggest that this complex functions as a dimeric motor. These studies, together with studies of yeast ISW2 imply that at least two types of ATP hydrolysis events accompany each cycle of nucleosome movement. We propose that ISWI motors may employ a kinetic proof reading type of mechanism to favor action on nucleosomes that are poised to be in condensed chromatin while inhibiting action on nucleosomes that are in fully active or fully condensed chromatin.
Bednar, Jan; Garcia-Saez, Isabel; Boopathi, Ramachandran; Cutter, Amber R; Papai, Gabor; Reymer, Anna; Syed, Sajad H; Lone, Imtiaz Nisar; Tonchev, Ognyan; Crucifix, Corinne; Menoni, Hervé; Papin, Christophe; Skoufias, Dimitrios A; Kurumizaka, Hitoshi; Lavery, Richard; Hamiche, Ali; Hayes, Jeffrey J; Schultz, Patrick; Angelov, Dimitar; Petosa, Carlo; Dimitrov, Stefan
Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures. Copyright © 2017 Elsevier Inc. All rights reserved.
Hinz, John M; Laughery, Marian F; Wyrick, John J
During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.
Le, Jenny V; Luo, Yi; Darcy, Michael A; Lucas, Christopher R; Goodwin, Michelle F; Poirier, Michael G; Castro, Carlos E
The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structural changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances, including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remain fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding but also significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.
Mittal, Chitvan; Blacketer, Melissa J; Shogren-Knaak, Michael A
The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.
Chung, Ho-Ryun; Dunkel, Ilona; Heise, Franziska; Linke, Christian; Krobitsch, Sylvia; Ehrenhofer-Murray, Ann E; Sperling, Silke R; Vingron, Martin
Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.
Andrews, Andrew J; Luger, Karolin
Chromatin is a highly regulated, modular nucleoprotein complex that is central to many processes in eukaryotes. The organization of DNA into nucleosomes and higher-order structures has profound implications for DNA accessibility. Alternative structural states of the nucleosome, and the thermodynamic parameters governing its assembly and disassembly, need to be considered in order to understand how access to nucleosomal DNA is regulated. In this review, we provide a brief historical account of how the overriding perception regarding aspects of nucleosome structure has changed over the past thirty years. We discuss recent technical advances regarding nucleosome structure and its physical characterization and review the evidence for alternative nucleosome conformations and their implications for nucleosome and chromatin dynamics.
Weicksel, Steven E; Xu, Jia; Sagerström, Charles G
Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.
Leonard, John D.; Narlikar, Geeta J.
SUMMARY The ATP-dependent chromatin assembly factor (ACF) is a dimeric motor that spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes. PMID:25684208
Landry, Joseph; Sharov, Alexei A.; Piao, Yulan; Sharova, Lioudmila V.; Xiao, Hua; Southon, Eileen; Matta, Jennifer; Tessarollo, Lino; Zhang, Ying E.; Ko, Minoru S. H.; Kuehn, Michael R.; Yamaguchi, Terry P.; Wu, Carl
We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo. PMID:18974875
Zhao, Xiujuan; Pei, Zhiyong; Liu, Jia; Qin, Sheng; Cai, Lu
In this work, a novel method was developed to distinguish nucleosome DNA and linker DNA based on increment of diversity combined with quadratic discriminant analysis (IDQD), using k-mer frequency of nucleotides in genome. When used to predict DNA potential for forming nucleosomes, the model achieved a high accuracy of 94.94%, 77.60%, and 86.81%, respectively, for Saccharomyces cerevisiae, Homo sapiens, and Drosophila melanogaster. The area under the receiver operator characteristics curve of our classifier was 0.982 for S. cerevisiae. Our results indicate that DNA sequence preference is critical for nucleosome formation potential and is likely conserved across eukaryotes. The model successfully identified nucleosome-enriched or nucleosome-depleted regions in S. cerevisiae genome, suggesting nucleosome positioning depends on DNA sequence preference. Thus, IDQD classifier is useful for predicting nucleosome positioning.
Takagi, Haruna; Inai, Yuta; Watanabe, Shun-ichiro; Tatemoto, Sayuri; Yajima, Mamiko; Akasaka, Koji; Yamamoto, Takashi; Sakamoto, Naoaki
The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin. To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity. Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator. Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region. Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC). These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity. Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity. These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator.
Rube, H Tomas; Song, Jun S
Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5' end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in ∼ 50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.
Raveh-Sadka, Tali; Levo, Michal; Segal, Eran
Transcriptional control is central to many cellular processes, and, consequently, much effort has been devoted to understanding its underlying mechanisms. The organization of nucleosomes along promoter regions is important for this process, since most transcription factors cannot bind nucleosomal sequences and thus compete with nucleosomes for DNA access. This competition is governed by the relative concentrations of nucleosomes and transcription factors and by their respective sequence binding preferences. However, despite its importance, a mechanistic understanding of the quantitative effects that the competition between nucleosomes and factors has on transcription is still missing. Here we use a thermodynamic framework based on fundamental principles of statistical mechanics to explore theoretically the effect that different nucleosome organizations along promoters have on the activation dynamics of promoters in response to varying concentrations of the regulating factors. We show that even simple landscapes of nucleosome organization reproduce experimental results regarding the effect of nucleosomes as general repressors and as generators of obligate binding cooperativity between factors. Our modeling framework also allows us to characterize the effects that various sequence elements of promoters have on the induction threshold and on the shape of the promoter activation curves. Finally, we show that using only sequence preferences for nucleosomes and transcription factors, our model can also predict expression behavior of real promoter sequences, thereby underscoring the importance of the interplay between nucleosomes and factors in determining expression kinetics. PMID:19451592
Raveh-Sadka, Tali; Levo, Michal; Segal, Eran
Transcriptional control is central to many cellular processes, and, consequently, much effort has been devoted to understanding its underlying mechanisms. The organization of nucleosomes along promoter regions is important for this process, since most transcription factors cannot bind nucleosomal sequences and thus compete with nucleosomes for DNA access. This competition is governed by the relative concentrations of nucleosomes and transcription factors and by their respective sequence binding preferences. However, despite its importance, a mechanistic understanding of the quantitative effects that the competition between nucleosomes and factors has on transcription is still missing. Here we use a thermodynamic framework based on fundamental principles of statistical mechanics to explore theoretically the effect that different nucleosome organizations along promoters have on the activation dynamics of promoters in response to varying concentrations of the regulating factors. We show that even simple landscapes of nucleosome organization reproduce experimental results regarding the effect of nucleosomes as general repressors and as generators of obligate binding cooperativity between factors. Our modeling framework also allows us to characterize the effects that various sequence elements of promoters have on the induction threshold and on the shape of the promoter activation curves. Finally, we show that using only sequence preferences for nucleosomes and transcription factors, our model can also predict expression behavior of real promoter sequences, thereby underscoring the importance of the interplay between nucleosomes and factors in determining expression kinetics.
Chen, Kaifu; Xi, Yuanxin; Pan, Xuewen; Li, Zhaoyu; Kaestner, Klaus; Tyler, Jessica; Dent, Sharon; He, Xiangwei; Li, Wei
Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome. PMID:23193179
In yeast and mammals, ATP-dependent chromatin remodeling complexes belonging to the SWI/SNF family play critical roles in the regulation of transcription, cell proliferation, differentiation and development. Homologs of conserved subunits of SWI/SNF-type complexes, including several putative ATPases and other core subunits, have been identified in plants. Here I summarize recent insights in structural organization and functional diversification of putative plant SWI/SNF-type chromatin remodeling complexes and discuss in a broader evolutionary perspective the similarities and differences between plant and yeast/animal SWI/SNF remodeling. I also summarize the current view of localization in nucleosome and dynamic behaviour in chromatin of linker (H1) histones and discuss significance of recent findings indicating that in both plants and mammals histone H1 is involved in determining patterns of DNA methylation at selected loci.
Celona, Barbara; Weiner, Assaf; Di Felice, Francesca; Mancuso, Francesco M.; Cesarini, Elisa; Rossi, Riccardo L.; Gregory, Lorna; Baban, Dilair; Rossetti, Grazisa; Grianti, Paolo; Pagani, Massimiliano; Bonaldi, Tiziana; Ragoussis, Jiannis; Friedman, Nir; Camilloni, Giorgio; Bianchi, Marco E.; Agresti, Alessandra
The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently