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

Epigenetics & Chromatin
Iyer Aditya MahadevanManchanahalli R Satyanarayana Rao

Abstract

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. 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 conf...Continue Reading

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Methods Mentioned

BETA
ELISA
ChIP
ChIP-seq
immunoprecipitation
pull down
co-IP
electrophoresis

Software Mentioned

FastQC
Perl scripts
ngsplot
Bowtie
R
MEME
house
Bowtie2
HOMER
MACS2

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