Abstract
BackgroundBiological relevance of the major testis specific histone H2B variant (TH2B) in sperm is not fully understood. Studies in TH2A/TH2B double knockout male mice indicate its role in chromatin compaction and male fertility. Additionally, the presence of TH2B and TH2A reportedly generates more dynamic nucleosomes, leading to an open chromatin structure characteristic of transcriptionally active genome. Given that mature human sperm are transcriptionally and translationally inactive, the presence of TH2B in mature sperm is intriguing. To address its role in sperm, we investigated the genome-wide localization of TH2B in sperm of fertile men.ResultsWe have identified the genomic loci associated with TH2B in fertile human sperm by ChIP-seq analysis. Bioinformatic analysis revealed ~ 5% sperm genome and 5527 genes to be associated with TH2B. Out of these 105 (1.9%) and 144 (2.6%) genes showed direct involvement in sperm function and early embryogenesis, respectively. Chromosome wide analysis for TH2B distribution indicated its least distribution on X and Y chromosomes and varied distribution on autosomes. TH2B showed relatively higher percentage of gene association on chromosome 4, 18, 3 and 2. TH2B enrichment was more in promoter and gene body region. Gene Ontology (GO) analysis revealed signal transduction and associated kinase activity as the most enriched biological and molecular function, respectively. We also observed the enrichment of TH2B at developmentally important loci, such as HOXA and HOXD and on genes required for normal sperm function, few of which were validated by ChIP-qPCR. The relative expression of these genes was altered in particular subgroup of infertile men showing abnormal chromatin packaging. Chromatin compaction positively correlated with sperm- motility, concentration, viability and with transcript levels of PRKAG2 and CATSPER B.ConclusionChIP-seq analysis of TH2B revealed a putative role of TH2B in sperm function and embryo development. Altered expression of TH2B associated genes in infertile individuals with sperm chromatin compaction defects indicates involvement of TH2B in transcriptional regulation of these genes in post meiotic male germ cells. This altered transcriptome may be a consequence or cause of abnormal nuclear remodeling during spermiogenesis.
Highlights
Spermatogenesis is a well synchronized and tightly regulated process by which male germ cells are formed
We have identified genomic loci associated with testis specific histone H2B variant (TH2B) in the sperm of fertile men by high-throughput sequencing, and association of a few genes was confirmed using Chromatin immunoprecipitation (ChIP)-Quantitative real time PCR (qPCR). qRT-Polymerase Chain Reaction (PCR) analysis was done to determine the relative abundance of transcripts of genes associated with TH2B in sperm from fertile- and infertile men and the observations corroborated with their chromatin compaction status
ChIP was performed using either anti-TH2B antibody or its Isotype control (IgG). 1.2 million sperm (1/10th of the cells used for ChIP) were micrococcal nuclease (MNase) digested, proteins were precipitated and used as positive control (Input)
Summary
Spermatogenesis is a well synchronized and tightly regulated process by which male germ cells are formed. Patankar et al Clin Epigenet (2021) 13:101 takes place in male germ cells It initiates with histone hyperacetylation followed by replacement of somatic histones with testis specific histone variants. Knockout studies with these variants testify their essential role in male infertility [1, 2]. Most of these variants are found to be involved in open chromatin structure formation.
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