Heterogeneity in the Epigenetic Landscape of Murine Testis-Specific Histone Variants TH2A and TH2B Sharing the Same Bi-Directional Promoter

Abstract

Testis-specific histone variants are crucial to promote open chromatin structure to enable nucleosome disassembly in the final stages of spermiogenesis. However, even after histone replacement, mature sperm retain a proportion of these variants, the function of which is unknown. The present study aimed to understand the functional relevance of the retained H2B and H2A variants, TH2B and TH2A. While no literature is available on the phenotype of TH2A knockouts, TH2B/TH2A double knockout male mice are reported to be infertile. In this study, ChIP-seq analysis was done for TH2B and TH2A to understand the epigenomics of the retained TH2B and TH2A, using murine caudal sperm. Distribution across genomic partitions revealed ∼35% of the TH2B peaks within ±5 kb of TSS whereas TH2A peaks distribution was sparse at TSS. Gene Ontology revealed embryo development as the most significant term associated with TH2B. Also, based on genomic regions, TH2B was observed to be associated with spindle assembly and various meiosis-specific genes, which is an important finding as TH2A/TH2B DKO mice have been reported to have defective cohesin release. A comparison of mouse and human TH2B-linked chromatin revealed 26% overlap between murine and human TH2B-associated genes. This overlap included genes crucial for embryogenesis. Most importantly, heterogeneity in the epigenetic landscape of TH2A and TH2B was seen, which is intriguing as TH2B and TH2A are well reported to be present in the same nucleosomes to promote open chromatin. Additionally, unlike TH2B, TH2A was enriched on the mitochondrial chromosome. TH2A was found to be associated with Nuclear insertion of Mitochondrial DNA sequences (NUMTs) in sperm. A comprehensive analysis of these observations indicates novel functions for the sperm-retained TH2B and TH2A.

Microhomology-Mediated Circular DNA Formation from Oligonucleosomal Fragments During Spermatogenesis

“…It is a common practice to re-use publicly-available genomics data generated in the same cell types for integrative analysis. Taking advantage of public ChIP-seq data for histones and their modifications in mouse sperm cells ( Jung et al, 2019; Jung et al, 2017; Singh et al, 2021 ), we found that eccDNAs were significantly enriched with certain histone variants and modifications (Figure 2C), and 7.46% of sperm eccDNAs in total were intersected with at least one ChlP-seq peaks. Considering that histones occasionally retained in sperms might not generate strong ChIP-seq signals exceeding the peak calling cutoff, a meta-gene analysis of ChIP-seq signals at and around sperm eccDNA regions will likely provide more insights.…”

“…Motif analysis and visualization were done by Two Sample Logo web server ( Vacic et al, 2006 ). Public ChIP-seq datasets ( Jung et al, 2019; Jung et al, 2017; Singh et al, 2021 ) were aligned to GRCm38 reference genome using Bowtie, and sorted and indexed using SAMtools version 1.7 ( Danecek et al, 2021 ). Picard MarkDuplicates version 2.18.14 was used to remove PCR duplicates.…”

Microhomology-mediated circular DNA formation from oligonucleosomal fragments during spermatogenesis

“…It is a common practice to reuse publicly available genomics data generated in the same cell types for integrative analysis. Taking advantage of public ChIP-seq data for histones and their modifications in mouse sperm cells ( Jung et al, 2019 ; Jung et al, 2017 ; Singh and Parte, 2021 ), we found that eccDNAs were significantly enriched with certain histone variants and modifications ( Figure 2C ), and 7.46% of sperm eccDNAs in total were intersected with at least one ChIP-seq peaks. Considering that histones occasionally retained in sperms might not generate strong ChIP-seq signals exceeding the peak calling cutoff, a meta-gene analysis of ChIP-seq signals at and around sperm eccDNA regions will likely provide more insights.…”

Insights into the sperm chromatin and implications for male infertility from a protein perspective

“…Histone variants and TPs also bear PTMs, which affects their binding affinity to DNA (Gupta et al, 2015(Gupta et al, , 2017Hazzouri et al, 2000;Kwak & Dohmae, 2016;Mishra et al, 2015;Pentakota et al, 2014;Pradeepa et al, 2009;Singh & Parte, 2021). Of note, TP2 acetylation reduces its ability to condense DNA (Pradeepa et al, 2009), suggesting that modified TPs may contribute to establish the final sperm epigenome.…”