Background TH2B is a major histone variant that replaces about 80–85% of somatic H2B in mammalian spermatocytes and spermatids. The post-translational modifications (PTMs) on TH2B have been well characterised in spermatocytes and spermatids. However, the biological function(s) of these PTMs on TH2B have not been deciphered in great detail. In our attempt to decipher the unique function(s) of histone variant TH2B, we detected the modification in the N-terminal tail, Serine 11 phosphorylation on TH2B (TH2BS11ph) in spermatocytes. Results The current study is aimed at understanding the function of the TH2BS11ph modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies with the highly specific antibodies revealed that TH2BS11ph histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body-associated proteins like Scp3, γH2AX, pATM, ATR, etc. Genome-wide occupancy studies as determined by ChIP sequencing experiments in P20 C57BL6 mouse testicular cells revealed that TH2BS11ph is enriched in X and Y chromosomes confirming the immunofluorescence staining pattern in the pachytene spermatocytes. Apart from the localisation of this modification in the XY body, TH2BS11ph is majorly associated with H3K4me3-containing genomic regions like gene promoters, etc. These data were also found to corroborate with the ChIP sequencing data of TH2BS11ph histone mark carried out in P12 C57BL6 mouse testicular cells, wherein we found the predominant localisation of this modification at H3K4me3-containing genomic regions. Mass spectrometry analysis of proteins that associate with TH2BS11ph-containing mononucleosomes revealed key proteins linked with the functions of XY body, pericentric heterochromatin and transcription. Conclusions TH2BS11ph modification is densely localised in the unsynapsed axes of the XY body of the pachytene spermatocyte. By ChIP sequencing studies in mouse P12 and P20 testicular cells, we demonstrate that TH2BS11ph is predominantly associated with H3K4me3 positive genomic regions like gene promoters, etc. We propose that TH2BS11ph modification could act alone or in concert with other histone modifications to recruit the appropriate transcription or XY body recombination protein machinery at specific genomic loci.

Various studies have focussed on understanding the repertoire and biological function of the post-translational modifications that occur on testis-specific histone variants like TH2B, Transition Proteins etc. In our attempt to decipher the unique functions of histone variant TH2B, we discovered a new modification Serine 12 phosphorylation on TH2B (TH2BS12P) in spermatocytes. Our present study is aimed at understanding the function of the TH2BS12P modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies revealed that TH2BS12P histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body axes associated proteins like Scp3, γH2AX, pATM, ATR etc. We also observe that TH2BS12P is associated with DSB initiator Spo11 and with several recombination related proteins like pATM, ATR, Rad51, γH2AX etc in vivo. This modification was also found to associate with transcription and recombination related histone marks like H3K4me3 and H3K36me3 in the context of mononucleosomes. Genome-wide occupancy studies as determined by ChIP sequencing experiments revealed that TH2BS12P is localised to subset of recombination hotspots, but majorly associated with H3K4me3 containing genomic regions like gene promoters. Mass spectrometry analysis of proteins that bind to TH2BS12P containing mononucleosomes revealed many proteins linked with the functions of pericentric heterochromatin, transcription and 3 / 88 recombination related pathways. We propose that TH2BS12P modification could act alone or in concert with other histone marks for recruitment of appropriate transcription or recombination protein machinery at specific genomic loci. This is the first report documenting the role of a post-translational modification of a germ cell specific histone variant in meiotic prophase I related events. / 88Recently, various studies have focussed on understanding the post-translational modifications on testis-specific histone variants like TH2B 33,51 , TP1 18 , TP2 18 , HILS1 40 , etc. During prophase I of meiosis, the homologous chromosomes synapse and undergo recombination at non-randomly selected loci. The exchange of genetic material is critical for the generation of diversity in the offspring. During leptotene interval, the global induction of Spo11 mediated DSBs occur 6 / 88 throughout the genome triggering the DNA damage response (DDR) 7,42 . Subsequently, MRN (Mre11-Rad50-Nbs1) complex recruits ATM kinase, catalyzes the first level of H2AX phosphorylation to form γH2AX 6,11,29 . The end resection and strand invasion mediated by MRN complex, RAD51, DMC1 and other proteins are characteristic of the next stage, the zygotene interval. During the pachytene stage, BRCA1 senses asynapsis and recruits ATR kinase for amplification of DDR signals along the unsynapsed axes for the establishment of the γH2AX domain in the XY body 8,60,77 . The region of homology between the X and the Y chromosomes termed as pseudo-autosomal region (PAR), is limited in size (~800 kb in mice) an...