scholarly journals Linker histone variant H1t is closely associated with repressed repeat-element chromatin domains in pachytene spermatocytes

2019 ◽  
Author(s):  
Iyer Aditya Mahadevan ◽  
Sanjeev Kumar ◽  
Manchanahalli R. Satyanarayana Rao
Keyword(s):  
Western Blot ◽  
Linker Histone ◽  
Histone Variant ◽  
Repeat Element ◽  
Open Chromatin ◽  
Gene Promoters ◽  
Chromatin Domains ◽  
Chip Sequencing ◽  
Pachytene Spermatocytes ◽  
Genomic Regions

AbstractBackgroundH1t is the major linker histone variant in pachytene spermatocytes, where it constitutes 50-60% of total H1. This linker histone variant was previously reported to localize in the nucleolar rDNA element in mouse spermatocytes. Our main aim was to determine the extra-nucleolar localization of this linker histone variant in pachytene spermatocytes.ResultsWe generated H1t-specific antibodies in rabbits and validated its specificity by multiple assays like ELISA, western blot, etc. Genome-wide occupancy studies, as determined by ChIP-sequencing in P20 mouse testicular cells revealed that H1t did not closely associate with active gene promoters and open chromatin regions. Annotation of H1t bound genomic regions revealed that H1t is depleted from DSB hotspots and TSS, but are predominantly associated with retrotransposable repeat elements like LINE and LTR in pachytene spermatocytes. These chromatin domains are repressed based on co-association of H1t observed with methylated CpGs and repressive histone marks like H3K9me3 and H4K20me3 in vivo. Mass spectrometric analysis of proteins associated with H1t-containing oligonucleosomes identified piRNA-PIWI pathway proteins, repeat-repression associated proteins and heterochromatin proteins confirming the association with repressed repeat-element genomic regions. We validated the interaction of key proteins with H1t-containing oligonucleosomes by use of ChIP-western blot assays. On the other hand, we observe majority of H1t peaks to be associated with the intergenic spacer of the rDNA element, also in association with SINE elements of the rDNA element. Thus, we have identified the genomic and chromatin features of both nucleolar and extranucleolar localization patterns of linker histone H1t in the context of pachytene spermatocytes.ConclusionsH1t-containing repeat-element LINE and LTR chromatin domains are associated with repressive marks like methylated CpGs, histone modifications H3K9me3 and H4K20me3, and heterochromatin proteins like HP1β, Trim28, PIWIL1 etc. Apart from localisation of H1t at the rDNA element, we demonstrate the extranucleolar association of this linker histone variant at repeat-associated chromatin domains in pachytene spermatocytes. We hypothesize that H1t might induce local chromatin relaxation to recruit heterochromatin and repeat repression-associated protein factors necessary for TE (transposable element) repression, the final biological effect being formation of closed chromatin repressed structures.

scholarly journals Linker histone variant H1t is closely associated with repressed repeat-element chromatin domains in pachytene spermatocytes

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Iyer Aditya Mahadevan ◽  
Sanjeev Kumar ◽  
Manchanahalli R. Satyanarayana Rao
Keyword(s):  
Linker Histone ◽  
Histone Variant ◽  
Repeat Element ◽  
Chromatin Domains ◽  
Pachytene Spermatocytes

Genome-wide profiling of histone 3 lysine 36 trimethylation in clear cell renal cell carcinoma.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 464-464
Author(s):  
Thai Huu Ho ◽  
Jeong-Heon Lee ◽  
Rafael Nunez Nateras ◽  
Erik P. Castle ◽  
Melissa L. Stanton ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Open Chromatin ◽  
Sequencing Analysis ◽  
Gene Desert ◽  
Cell Renal Cell Carcinoma ◽  
Chip Sequencing ◽  
Genome Wide ◽  
A Genome ◽  
Genomic Regions

464 Background: Although the von Hippel-Lindau (VHL) tumor suppressor gene is mutated in 60% of ccRCC, deletion of VHL in mice is insufficient for tumorigenesis. Sequencing of ccRCC tumors identified mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. We hypothesize that loss of SETD2 methyltransferase activity alters the genome wide pattern of H3K36 trimethylation (H3K36me3) in ccRCC, and contributes to the cancer phenotype. Methods: To generate a genome-wide profile of H3K36me3 in frozen nephrectomy samples and RCC cell lines, we optimized a chromatin immunoprecipitation (ChIP) protocol for the isolation of DNA associated with H3K36me3. H3K36me3 is associated with open chromatin and an H3K36me3-specific antibody was used for immunoprecipitation of endogenous H3K36me3-bound DNA. ChIP PCR primers were optimized for active genes, such as actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a “gene desert” on chromosome 12 (negative control). ChIP libraries were then generated from 3 paired uninvolved kidney and RCC and 2 RCC cell lines. In order to identify H3K36Me3 upregulated regions in uninvolved kidney and RCC, reads from the ChIP sequencing were mapped to the human genome using Burrows-Wheeler Aligner and SICER algorithms. Results: Using ChIP PCR, we found that active genomic regions were enriched 15-30 fold over the negative controls indicating that the quality and yield of immunoprecipitated DNA/chromatin complexes from frozen tissue was sufficient for ChIP sequencing. A preliminary ChIP sequencing analysis of RCC cell lines and frozen ccRCC tissue indicates that H3K36me3 enriched DNA sequences were mapped to exons (31.3%) compared to introns (13.5%, p<0.001), consistent with the role of H3K36me3 in transcription. Conclusions: Genomic regions enriched for H3K36Me3 binding were identified from patient-derived tissue and RCC cell lines. Current efforts are focused on comparing the H3K36me3 profiles between matched tumor and uninvolved kidney ChIP libraries to generate a genome wide map of dysregulated H3K36me3 modifications.

scholarly journals Quantitative modeling of fine-scale variations in the Arabidopsis thaliana crossover landscape

2021 ◽  
Author(s):  
Yu-Ming Hsu ◽  
Matthieu Falque ◽  
Olivier Martin
Keyword(s):  
Arabidopsis Thaliana ◽  
Contextual Information ◽  
Quantitative Modeling ◽  
Open Chromatin ◽  
Gene Promoters ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Homologous Sequences ◽  
Experimental Variation ◽  
Genomic Regions

In essentially all species where meiotic crossovers have been studied, they occur preferentially in open chromatin, typically near gene promoters and to a lesser extent at the end of genes. Here, in the case of Arabidopsis thaliana, we unveil further trends arising when one considers contextual information, namely summarized epigenetic status, size of underlying genomic regions and degree of divergence between homologs. For instance we find that intergenic recombination rate is reduced if those regions are less than 1.5 kb in size. Furthermore, we propose that the presence of single nucleotide polymorphisms is a factor driving enhanced crossover rate compared to when homologous sequences are identical, in agreement with previous works comparing rates in homozygous and heterozygous blocks. Lastly, by integrating these different factors, we produce a quantitative and predictive model of the recombination landscape that reproduces much of the experimental variation.

scholarly journals Genome-Wide Occupancy Reveals The Localization of H1T2 (H1fnt) To Repeat Regions and A Subset of Transcriptionally Active Chromatin Domains in Mammalian Spermatids

2020 ◽  
Author(s):  
Shalini Vasantha ◽  
Utsa Bhaduri ◽  
Anjhana C Ravikkumar ◽  
Anusha Rengarajan ◽  
Satyanarayana Rao
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Domain Architecture ◽  
Linker Histone ◽  
Mass Spectrometry Analysis ◽  
Open Chromatin ◽  
Active Chromatin ◽  
Chromatin Domains ◽  
Protein Partners ◽  
Genome Wide

Abstract Background: H1T2/H1FNT is a germ cell specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone to protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown.Results: Sequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions (promoter-TSS, exons and introns) of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2 associated chromatin mainly as nucleo-skeleton components, RNA binding proteins and chaperones.Conclusions: Linker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids.

scholarly journals Genome-wide occupancy reveals the localization of H1T2 (H1fnt) to repeat regions and a subset of transcriptionally active chromatin domains in rat spermatids

2020 ◽  
Author(s):  
Shalini Vasantha ◽  
Utsa Bhaduri ◽  
Anjhana C Ravikkumar ◽  
Anusha Rengarajan ◽  
Satyanarayana Rao
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Domain Architecture ◽  
Linker Histone ◽  
Mass Spectrometry Analysis ◽  
Open Chromatin ◽  
Active Chromatin ◽  
Chromatin Domains ◽  
Protein Partners ◽  
Genome Wide

Abstract Background H1T2/H1FNT is a germ cell specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone to protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown.Results Sequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions (promoter-TSS, exons and introns) of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2 associated chromatin mainly as nucleo-skeleton components, RNA binding proteins and chaperones.Conclusions Linker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids.

scholarly journals Rat PRDM9 shapes recombination landscapes, duration of meiosis, gametogenesis, and age of fertility

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Ondrej Mihola ◽  
Vladimir Landa ◽  
Florencia Pratto ◽  
Kevin Brick ◽  
Tatyana Kobets ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Hybrid Sterility ◽  
Strand Break ◽  
Reproductive Age ◽  
Open Chromatin ◽  
Gene Promoters ◽  
Functional Regions ◽  
Crossover Rate ◽  
Specific Hybrid ◽  
Pachytene Spermatocytes

AbstractBackgroundVertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mousePrdm9gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear.ResultsWe introducedPrdm9deletions into theRattus norvegicusgenome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-typePrdm9allele shared 88% hotspots but strains with differentPrdm9alleles only 3%. AfterPrdm9deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding toPrdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility,Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mousePrdm9mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants.ConclusionsWe hypothesize that the relative increased fertility of rat versus mousePrdm9mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation—spermatid development and reproductive age—that may help to explain male-specific hybrid sterility.

scholarly journals Genome-wide occupancy reveals the localization of H1T2 (H1fnt) to repeat regions and a subset of transcriptionally active chromatin domains in rat spermatids

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Vasantha Shalini ◽  
Utsa Bhaduri ◽  
Anjhana C. Ravikkumar ◽  
Anusha Rengarajan ◽  
Rao M. R. Satyanarayana
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Domain Architecture ◽  
Linker Histone ◽  
Mass Spectrometry Analysis ◽  
Open Chromatin ◽  
Active Chromatin ◽  
Chromatin Domains ◽  
Protein Partners ◽  
Genome Wide

Abstract Background H1T2/H1FNT is a germ cell-specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone-to-protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown. Results Sequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome-wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions (promoter-TSS, exons and introns) of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2-associated chromatin mainly as nucleo-skeleton components, RNA-binding proteins and chaperones. Conclusions Linker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids.

scholarly journals 3D reconstruction of genomic regions from sparse interaction data

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Julen Mendieta-Esteban ◽  
Marco Di Stefano ◽  
David Castillo ◽  
Irene Farabella ◽  
Marc A Marti-Renom
Keyword(s):  
3D Models ◽  
Genomic Region ◽  
Gene Promoters ◽  
Chromosome Conformation ◽  
The Matrix ◽  
Chromatin Structural ◽  
A Cell ◽  
Similar Accuracy ◽  
Genomic Regions ◽  
Interaction Matrices

Abstract Chromosome conformation capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterize short- and long-range enhancer–promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organization from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalization, detection of significant interactions and reconstruction of the full 3D organization of the genomic region despite of the data sparseness. Specifically, it builds, with as low as the 2–3% of the data from the matrix, reliable 3D models of similar accuracy of those based on dense interaction matrices. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organizational features such as the formation of different networks of active gene communities.

scholarly journals The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pierre Bourguet ◽  
Colette L. Picard ◽  
Ramesh Yelagandula ◽  
Thierry Pélissier ◽  
Zdravko J. Lorković ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone H1 ◽  
Epigenetic Modifications ◽  
Flowering Plants ◽  
Linker Histone ◽  
Histone Variant ◽  
Null Mutant ◽  
Chromatin Compaction ◽  
Linker Histone H1

AbstractIn flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

scholarly journals Mechanisms of Nucleosome Reorganization by PARP1

2021 ◽  
Vol 22 (22) ◽  
pp. 12127
Author(s):  
Natalya V. Maluchenko ◽  
Dmitry K. Nilov ◽  
Sergey V. Pushkarev ◽  
Elena Y. Kotova ◽  
Nadezhda S. Gerasimova ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Dna Repair ◽  
Gene Regulation ◽  
Transcription Initiation ◽  
Histone H1 ◽  
Linker Histone ◽  
Gene Promoters ◽  
Linker Histone H1 ◽  
Nucleosomal Dna ◽  
Dna Ends

Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair, chromatin organization and transcription. During transcription initiation, PARP1 interacts with gene promoters where it binds to nucleosomes, replaces linker histone H1 and participates in gene regulation. However, the mechanisms of PARP1-nucleosome interaction remain unknown. Here, using spFRET microscopy, molecular dynamics and biochemical approaches we identified several different PARP1-nucleosome complexes and two types of PARP1 binding to mononucleosomes: at DNA ends and end-independent. Two or three molecules of PARP1 can bind to a nucleosome depending on the presence of linker DNA and can induce reorganization of the entire nucleosome that is independent of catalytic activity of PARP1. Nucleosome reorganization depends upon binding of PARP1 to nucleosomal DNA, likely near the binding site of linker histone H1. The data suggest that PARP1 can induce the formation of an alternative nucleosome state that is likely involved in gene regulation and DNA repair.

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