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

Abstract

Background: H1t 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. Results: We 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. Conclusions: H1t-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 localization 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.

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

“…Numerous studies have identified H1T2 as a critical testes-specific histone H1 for spermiogenesis, lack of which results in reduced fertility because of delayed nuclear condensation and aberrant elongation characterized by acrosome detachment and fragmented DNA [ 22 ]. With this background, we were curious to gain more insights into the genomic functions of H1T2 in the context of our recent studies on the other two testes-specific histone variants, H1t and HILS1 [ 24 , 25 ].

Fig.

…”

“…Earlier studies from our laboratory had analyzed the epigenetic status of the LINE repeats at different stages of spermatogenesis in the context of linker histones occupancy. In pachytene spermatocytes, H1t decorates the methylated LINE and LTR repeats in addition to heterochromatin repressive marks [ 25 ]. As the sperm differentiation progresses through histone hyper acetylation and incorporation of other histone variants, HILS1 gets incorporated to provide a more loosened chromatin to facilitate the histone replacement, still maintaining the repressed chromatin architecture of the majorly bound LINE repeats [ 24 ].…”

“…An interesting observation from this study is the association of H1T2 to a subset of promoter-TSS chromatin domains in spermatids (~ 3% of total CHIP-seq reads). In contrast, only 0.6 and 0.08% of the ChIP-seq reads were associated with promoter regions by H1t and HILS1, respectively [ 24 , 25 ]. Detailed annotation of the H1T2 bound LINE repeats revealed the major association to a particular class of LINE repeat, LINE L1 Rn (Fig.…”

“…More recently, focused studies have been initiated to understand how the sequence diversity of H1 proteins leads to distinct chromatin structures associated with different functional properties. Studies from our laboratory recently focused on the mechanism by which testes-specific H1 variants contribute to diverse chromatin function by studying their genomic localization, PTMs and chromatin loci specific interacting proteins [ 24 , 25 ]. Linker histone H1t constituting about 50–60% of total H1 content induces local chromatin relaxation to recruit the appropriate protein machinery for establishing closed chromatin repressed structures [ 25 ].…”

“…Studies from our laboratory recently focused on the mechanism by which testes-specific H1 variants contribute to diverse chromatin function by studying their genomic localization, PTMs and chromatin loci specific interacting proteins [ 24 , 25 ]. Linker histone H1t constituting about 50–60% of total H1 content induces local chromatin relaxation to recruit the appropriate protein machinery for establishing closed chromatin repressed structures [ 25 ]. An earlier study from our laboratory had shown that HILS1, whose expression is mainly confined to the elongating spermatids is involved in the maintenance of repressed chromatin architecture by preferential binding to LINE element containing chromatin domains in rat spermatids.…”