Histone H1 limits DNA methylation in Neurospora
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ABSTRACT:
ORGANISM(S): Neurospora crassa
PROVIDER: GSE76982 | GEO | 2016/04/20
SECONDARY ACCESSION(S): PRJNA309184
REPOSITORIES: GEO
Similar Datasets
Project description:Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the [Formula: see text]hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the [Formula: see text]hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.
| S-EPMC4938642 | biostudies-literature
Project description:Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-FLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stochiometery of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in wildtype and the ∆hH1 strain followed by sequencing (MNase-Seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation using methylC-sequencing (mC-Seq) revealed a modest but global increase in DNA methylation in the ∆hH1 mutant. Together, these data suggest that H1 acts as a non-specific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.
2016-04-20 | GSE78157 | GEO
Project description:In 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 an 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.
2021-03-26 | GSE146948 | GEO
Project description:DNA methylation and histone H1 mediate transcriptional silencing of genes and transposable elements, but how they interact is unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. Here we show that H1 is enriched in methylated sequences, including genes, of Arabidopsis thaliana, yet this enrichment is independent of DNA methylation. Loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. Hypomethylation of genes also activates antisense transcription, which is enhanced by H1 loss. Our results demonstrate that H1 and DNA methylation jointly maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.
2019-11-20 | GSE122394 | GEO
Project description:We studied how H1 regulates higher-order chromatin architecture by performing Hi-C. In addition, we tested the effects of H1 domains on nucleosome organisation and DNA methylation by performing MNase-seq and Bisulfite-seq.
2024-02-15 | GSE176526 | GEO
Project description:Functional genomic states are maintained by reinforcing chromatin interactions that exclude the components of other states. Plant heterochromatin features methylation of histone H3 at lysine 9 (H3K9me) and extensive DNA methylation. However, DNA methylation is also catalyzed by a mostly euchromatic small RNA-directed pathway (RdDM) thought to seek H3K9me. How RdDM is excluded from H3K9me-rich heterochromatin is unclear. Here we show that without histone H1, RdDM enters heterochromatin, preferentially at nucleosome linker DNA. Surprisingly, this does not require SHH1, the RdDM component that binds H3K9me. Furthermore, H3K9me is dispensable for RdDM, as is CG DNA methylation. Instead, we find that non-CG methylation is specifically required for small RNA biogenesis, and without H1 small RNA production quantitatively expands to non-CG methylated loci. Our results demonstrate that H1 enforces the separation of euchromatic and heterochromatic DNA methylation pathways by excluding the small RNA-generating branch of RdDM from non-CG methylated heterochromatin.
2021-12-01 | GSE179796 | GEO
Project description:Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTM). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes for four canonical histones and five histone variants. By contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of tgh1-like does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutating post-translationally modified amino acids results in defects in cell division like ∆tgh1-like, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii.
2022-11-30 | PXD037318 | Pride
Project description:Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ~147 bp of DNA wrapped ~1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ~160 bp, and then converts it to a core particle, containing ~147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these "proto-chromatosomes" are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.
2015-09-23 | GSE65889 | GEO
Project description:Within the cell nucleus, histone H1 is the most mobile histone. Yet, it is regarded as key to the establishment and stability of chromatin higher-order structure. The implication is that chromatin higher-order structure is dynamic, in part due to the mobility of H1. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq) is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure. A second formaldehyde fixation after antibody binding to intact in situ chromatin, but before sonication (xxChIP-seq), preserves this information and reveals which histone epitopes are inaccessible in situ. In this study, the distribution of two histone H1 variants H1.2 and H1.5 is defined and compared by both xChIP-seq and xxChIP-seq in undifferentiated HL-60/S4 cells, illustrating the influences of preserved chromatin higher-order structure. We have found that xChIP and xxChIP signals are anticorrelated. H1.2 versus H1.5-enrichments were particularly distinct near bound Pol II, SMC3 proteins, as well as domains marked by H3K4me1, H3K9ac, H3K27ac and H3K36me3. H1.5-enriched regions have an average nucleosome repeat length NRL=184 bp, as opposed to 190 bp for H1.2-enriched regions. We have also studied the distribution of H1 variants with respect to DNA methylation. Most changes of DNA methylation during differentiation appear within ~60bp from the nucleosome entry/exit and are preferably associated with H1.2-bound nucleosomes, whereas H1.5 histones are depleted from CpGs. Our results suggest that different physical packing of nucleosomes may exist in H1.2- versus H1.5-enriched areas, characterized by differential H1 epitope exposure as assessed by xChIP and xxChIP.
2020-06-14 | GSE136264 | GEO
Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.
2010-06-21 | E-GEOD-16884 | biostudies-arrayexpress