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Th2bs11ph Histone Mark Is Enriched in the Unsynapsed Axes of the XY Body and Predominantly Associates with H3k4me3-Containing Ge

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Th2bs11ph Histone Mark Is Enriched in the Unsynapsed Axes of the XY Body and Predominantly Associates with H3k4me3-Containing Ge Mahadevan et al. Epigenetics & Chromatin (2019) 12:53 https://doi.org/10.1186/s13072-019-0300-y Epigenetics & Chromatin RESEARCH Open Access TH2BS11ph histone mark is enriched in the unsynapsed axes of the XY body and predominantly associates with H3K4me3-containing genomic regions in mammalian spermatocytes Iyer Aditya Mahadevan1†, Satyakrishna Pentakota2†, Raktim Roy3, Utsa Bhaduri1 and Manchanahalli R. Satyanarayana Rao1* Abstract Background: TH2B is a major histone variant that replaces about 80–85% of somatic H2B in mammalian spermato- cytes and spermatids. The post-translational modifcations (PTMs) on TH2B have been well characterised in spermato- cytes 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 modifcation 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 modifcation in the context of processes that occur during meiotic prophase I. Immunofuorescence studies with the highly specifc 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 confrming the immunofuorescence staining pattern in the pachytene spermatocytes. Apart from the localisation of this modifcation 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 TH2B- S11ph histone mark carried out in P12 C57BL6 mouse testicular cells, wherein we found the predominant localisation of this modifcation 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, pericen- tric heterochromatin and transcription. Conclusions: TH2BS11ph modifcation 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 modifcation could act alone or in concert with other histone modifcations to recruit the appropriate transcription or XY body recombination protein machinery at specifc genomic loci. *Correspondence: [email protected] †Iyer Aditya Mahadevan and Satyakrishna Pentakota contributed equally to this work 1 Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientifc Research, Jakkur PO., Bangalore 560064, India Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mahadevan et al. Epigenetics & Chromatin (2019) 12:53 Page 2 of 23 Keywords: TH2B serine 11 phosphorylation, Spermatocytes, TH2B, Immunofuorescence, Coimmunoprecipitation, H3K4me3 co-association Background nucleosomes. Histone PTMs are key molecular play- Mammalian spermatogenesis ofers an excellent model ers in epigenomic functions [17, 18]. Recently, various system to study chromatin remodelling by histone vari- studies have focussed on understanding the post-trans- ants as the testis is known to express a large number of lational modifcations on testis-specifc histone variants core and linker histone variants in a stage-specifc man- like TH2B [16, 19], TP1 (Transition Protein 1) [20], TP2 ner [1–6]. It is hypothesised that chromatin locus-spe- (Transition Protein 2) [20], HILS1 (Histone Linker H1 cifc histone replacement with histone variants could Spermatid specifc 1) [21], etc. be a possible basis for genome reprogramming in germ During prophase I of meiosis, the homologous chro- cells. Testis-specifc histone variants play critical roles mosomes synapse and undergo recombination at non- during the germ cell development. H3t (testis-specifc randomly selected loci. Te exchange of genetic material histone variant of H3) is essential for the process of is critical for the generation of diversity in the ofspring. spermatogonial diferentiation and ensures entry into During leptotene interval, the global induction of Spo11- meiosis [7]. Te loss of TH2A and TH2B leads to male mediated DSBs occurs throughout the genome triggering sterility with defects in cohesin release during interki- the DNA damage response (DDR) [22, 23]. Subsequently, nesis and histone to protamine replacement, suggesting MRN (Mre11-Rad50-Nbs1) complex recruits ATM an essential role of the testis-specifc histone variants kinase, and catalyses the frst level of H2AX phospho- during critical periods of male germ development [8]. rylation to form γH2AX [24–26]. Te end resection and TH2B, a synonym of germ cell-specifc H2B.1 (or strand invasion mediated by MRN complex, RAD51, TS H2B.1) [9] was one of the earliest histone vari- DMC1 and other proteins are characteristic of the next ants discovered in mammalian testis [10, 11]. To date, stage, the zygotene interval. During the pachytene stage, it is the only testis-specifc histone variant known to BRCA1 senses asynapsis and recruits ATR kinase for replace a core histone on a genome-wide scale replac- amplifcation of DDR signals along the unsynapsed axes ing 80–85% of H2B in spermatocytes and spermatids for the establishment of the γH2AX domain in the XY [12]. Te pachytene nucleosome core particle harbour- body [27–29]. Te region of homology between the X and ing the TH2B molecule was shown to be less compact the Y chromosomes termed as pseudo-autosomal region compared to H2B-containing nucleosome core par- (PAR) is limited in size (~ 800 kb in mice) and is largely ticle [13, 14]. hTSH2B (human TH2B)-reconstituted unsynapsed during meiosis. Terefore, to ensure chro- histone octamer was found to be less stable than the mosome segregation with at least one crossover in PAR, H2B-reconstituted histone octamer in vitro [15]. On a higher crossover density and increased DSB occurrence the other hand, the loss of mouse TH2B is compen- are observed in the PAR compared to that of autosomes sated for upregulation of H2B and compensatory his- [30]. Te increase in DSB sites is caused by specialised tone modifcations on the core histones H2B, H3 and chromatin confguration in PAR where DNA is organised H4 in germ cells. However, the expression of tagged- on a longer axis with shorter chromatin loops compared TH2B protein created a dominant negative phenotype, to autosomes [31, 32]. Te non-PAR regions of the X resulting in a defective histone to protamine replace- and Y chromosomes are transcriptionally silenced dur- ment that led to male sterility [12]. TH2B shares 85% ing meiotic prophase by a process termed as meiotic sex sequence similarity with canonical histone H2B with majority of the diferences being at its N-terminus. We surmised that these diferences or the post-transla- Table 1 List of datasets used for computational data tional modifcations acquired by some of the residues analyses could contribute to unique functions of TH2B. In this direction, Pentakota et al. [16] characterised the rep- Dataset Geo accession IDs ertoire of post-translational modifcations (PTMs) on DSB hotspots GSE93955 histone variant TH2B isolated from spermatocytes and TSS (of mouse) GENCODE spermatid stages. By computational analysis, it was also H3K4me3 GSE35498 shown that the amino acid diferences and the post- H3K4me3 common GSE93955 translational modifcations acquired by some of the H3K4me3 (B6 specifc) GSE93955 residues cause the destabilisation of TH2B-containing TH2B GSE45915 Mahadevan et al. Epigenetics & Chromatin (2019) 12:53 Page 3 of 23 chromosome inactivation (MSCI) [33, 34]. Te crossover terminal tail (Fig. 1b, c). In an attempt to decipher vari- products are generated at the end of pachytene [35]. Te ous PTMs on TH2B, we purifed in vivo TH2B from completion of meiosis I yields secondary spermatocytes 30-day rat testicular cells by the reverse-phase HPLC which undergo meiotic II division to produce haploid technique. By employing a diferent set of procedures round spermatids. that includes enzyme digestion and post-mass spec- In our eforts to gain insight into the unique func- trometry analyses of various PTMs obtained on in vivo tions of TH2B particularly during meiotic prophase I, TH2B, we detected the modifcation TH2BS11ph we detected a post-translational modifcation in the (TH2B serine 11 phosphorylation) in spermatocytes. amino terminal end that was already reported in another Tis modifcation was already detected in TH2B from study in round spermatid TH2B [36], Serine 11 phos- round spermatids by Luense
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