H1FNT/H1T2 – a highly divergent germ cell specific linker histone
Similar to all metazoan linker histones, H1T2 also possess a short, flexible N-terminal tail domain (NTD), a central globular domain (GD) with a winged-helix motif (three α helix) and a highly extended C-terminal tail domain (CTD). Figure 1A summarizes the comparison of H1T2 sequences from different mammalian species. A comparison of sequences of H1T2 with H1.4 (somatic) or H1t (germ cell specific) reveals that H1T2 is more diverged (≤ 21% similarity) from other linker histones. The human orthologue of H1T2 has highly diverged from rodents, with only 37.6% homology between mouse and human H1T2 sequence, 38.4% similarity between rat and human H1T2, while sharing most similarity with the gorilla orthologue with 97.6% identity. The highly extended C-terminal domain of H1T2 consists of a Walker motif (ATP/GTP binding), SR domain with SR repeats and many phosphorylation sites (Fig 1B). At the same time, H1T2 proteins lack the 16-mer (S/TPKK) motif, which is known to have DNA condensation properties (26). In the globular domain, human H1T2 shares aminoacid identity at several positions with the rodent specific H1T2, whereas the ATP binding motif and extended C-terminus including the SR domain are not present (Fig 1B). It is interesting to note that while the general property of the C-terminal domain of linker histone is to stabilize higher order chromatin folding, functional sub domains which are present in the CTD of rodent specific H1T2 are absent in higher mammals. Comparative study of testis protein evolution in rodents identified H1T2 as one of the rapidly evolving testis-expressed proteins (27). H1T2 possesses a putative winged helix domain consisting three alpha helices in addition to a unique coiled coil domain in the C-terminal domain (Fig 1C & D). A similar domain is reported in the N-terminal domain of Drosophila linker histone Mst77f, which is responsible for the DNA aggregation property of the H1 (28). Direct alignment of Mst77f with either HILS1 or H1T2 failed to uncover any significant sequence conservation with less than 15% identity, even though HILS1 has been referred in the literature as a putative mammalian homologue of Mst77f (28). Extensive studies on Drosophila MSt77f revealed dual roles in spermatogenesis, firstly involved in regional distribution of chromatin beneath the stripe of microtubules in histone-based nuclei, and secondly, it is essential for nuclear shaping through the stabilization of microtubules (9, 28). We found a high degree of similarity between Mst77f and H1T2 proteins with respect to coiled coil domains. Numerous studies have identified H1T2 as a critical testes specific histone H1 for spermiogenesis, lack of which results in reduced fertility because of delayed nuclear condensation and aberrant elongation characterized by acrosome detachment and fragmented DNA (22). With this background, we were curious to gain more insights into the genomic functions of H1T2 in the context of our recent studies on the other two testes specific histone variants, H1t and HILS1 (24, 25).
Characterization of antibody against CTD of rat H1T2 protein
Since H1T2 has been shown to occupy specialized chromatin regions in the apical region of spermatids, we wanted to analyze the genomic domains bound to H1T2 within these regions by ChIP-sequencing analysis. Because of the unavailability of a ChIP grade commercial antibody against the linker histone H1T2, we raised in house antibody against the C-terminal domain of rat H1T2, using the synthetic peptide (303-EQQYVSAKEQEYVRTKEQEC-321) (Fig 1B), which has shown earlier to be a strong immunogen (22). As expected, a strong immunoreactive signal was observed with dot blot analysis using the peptide as an antigen when probed with the affinity purified antibody (Fig 2A). Western blotting analysis was performed with the tissue lysate from both liver and testes of 35-50 days old rats. The antibodies reacted strongly with the testicular lysate with a distinct band at 54Kda. In contrast, the antibody failed to recognize any of the somatic H1 subtypes in the liver lysate, confirming the specificity of the antibody (Fig 2B). There was no signal observed in the peptide competition control lane, wherein the antibody was pre-incubated with 200-fold molar excess of the immunogen. H3 antibody was used as a positive control in these experiments, wherein we observed a positive signal for both liver and testes lysate. Western blot analysis was also performed with 25 and 50 day old rat testes acid extracts, wherein a strong H1T2 signal was observed with the 50day old rat testes extract (contributed predominanantly by round/elongnating spermatids). 10 day old rat testes lysate acted as a negative control for the experiment, which were contributed predominantly by somatic cells and spermatogonia (Fig 2C). Taken together, these experiments confirm that the antibody raised against the C terminal domain of rat H1T2 reacts specifically with the germ cell specific linker histone H1T2, and does not react with any other linker histone variants. Additionally, H1T2 signals were not detected in the histone extracts of mature sperm collected from rat epididymis (Fig 2D). Further immunolocalization studies of H1T2 in round and elongating spermatids revealed typical localization pattern with a cap like structure in the polar region of round spermatids, agreeing with the already established localization pattern of H1T2 signal at the nuclear region beneath the acrosome (Fig 2 E) (29).
H1T2 binds to distinct genomic domains in round/elongating spermatids
ChIP-sequencing was performed using the highly specific H1T2 antibody in rat round/elongating spermatids chromatin. Chromatin samples were collected from 35-50 day old rat testes (with a major population being represented by round and elongating spermatids). Cross linked chromatin was subjected to sonication for 45 cycles (15 sec on/15 sec off cycle), followed by immunoprecipitation using H1T2 antibody. Western blot analysis confirmed the immunoprecipitation with a positive signal at 54 KDa both in the input and IP lanes, where as the antibody was unable to recognize any protein in the peptide competition lane (antibody was competed with ~200 fold molar excess of the immunogen). Non specific IgG also acted as a negative control for the H1T2 immunoprecipitation experiment (Fig 2F). H1T2 immunoprecipitated DNA from the soluble extract of rat round/elongating spermatids was subjected to next-generation sequencing using Illumina HiSeq X Ten system using paired-end reads of 150nt length. After preprocessing the raw reads, the clean reads were aligned against UCSC Rattus norvegicus genome (rn6) using Bowtie2 (version 2.2.3) (Fig 3A). To analyze the extent of H1T2 occupancy in the spermatid genome, genomic regions associated to H1T2 was determined by MACS1.4.2 software with a p value cutoff of 1e-05. A total of 11570 enriched regions were obtained for H1T2 and the chromosome wise plots were generated (Fig 3B; Additional file 1: Table S1). The number of regions bound to H1T2 is maximally associated with chromosome 1 and 2, whereas the remaining peaks were evenly distributed across other chromosomes (Fig 3C; Additional file 2: Table S2). We have also analyzed the peak length distribution and fold enrichment across different chromosomes and the data is represented in the supplementary section (Additional file 3 & 4). To analyze the distribution of H1T2 peaks in various genomic domains of rat spermatid genome, H1T2 enriched genomic regions were annotated and categorized into CpG islands, repetitive elements, exons, introns, intergenic regions, 3′UTR and 5′UTR using HOMER v4.7 (Fig 3D). H1T2 enriched regions showed a bias towards the intergenic regions (71.56%), while at the same time a significant number of peaks were in the genic regions (promoter (2.94%), 5′ UTR (0.47%), 3′ UTR (0.22%), exon (2.43%), intron (19.27%). Next the repeat density in the H1T2 associated intergenic regions was calculated separately for interspersed repeat types including LINEs and LTRs (long autonomous retrotransposons) and SINEs (nonautonomous short <300 bp retrotransposons that co-opt the LINE transposition machinery) (30). The peak density is much lower in SINE/LTR regions compared to LINE rich region, with around 66% of H1T2 bound intergenic regions being LINE repeats (Fig 3E; Additional file 5:Table S5). Traditional views considered spermatozoa as transcriptionally silent cells, especially LINE repeats as the successful retrotranspososn which is epigenetically repressed by CpG DNA methylation in conjunction with the piRNA pathway with multiple epigenetic mechanisms (31). Earlier studies from our laboratory had analyzed the epigenetic status of the LINE repeats at different stages of spermatogenesis in the context of linker histones occupancy. In pacheytene spermatocytes, H1t decorates the methylated LINE and LTR repeats in addition to heterochromatin repressive marks (25). As the sperm differentiation progresses through histone hyper acetyaltion and incorporartion of other histone variants, HILS1 gets incorporated to provide a more loosened chromatin to facilitate the histone replacement, still maintaining the repressed chromatin architecture of the majorly bound LINE repeats (24). An interesting observation from this study is the association of H1T2 to a subset of promoter-TSS chromatin domains in spermatids (~3% of total CHIP-seq reads). In contrast, only 0.6% and 0.08% of the ChIP-seq reads were associated with promoter regions by H1t and HILS1 respectively (24, 25). Detailed annotation of the H1T2 bound LINE repeats revealed the major association to a particular class of LINE repeat, LINE L1 Rn (Fig 3F; Additional file 6: Table S6) similar to our previous observation with respect to HILS1 (24). Full-length rat L1s (L1Rn) with intact open reading frames (ORFs) is considered as the functional master copies for retrotransposition. In addition, a recent study has reported strong L1Rn RNA expression and hypomethylation status in rat testes (32). By using the motif analysis program MEME-ChIP, we identified three consensus motifs to be significant in the total H1T2 peaks (Fig 3G; Additional file 7 Table S7) and the percentage of occurrence of each of the motif were found to be 55.32% (Motif A), 30.54% (Motif B) and 41.95% (Motif C).
Functional annotation of the H1T2 associated genomic domain of spermatid genome
It was most surprising that, ChIP-sequencing analysis identified 321 gene promoters as the potential binding sites for H1T2 predominantly between (+/-) 3Kb from transcription start site in rat round/elongating spermatids. When we carried out distribution analysis of our ChIP peaks within the +/-3kb region centered around TSS of genes, the distribution pattern turned into a sharp bell shaped curve, showing a peak at the TSS position (Fig 4A). To gain insights into these regions, we carried out gene ontology analysis of the genes associated with these promoters (Fig 4B; Additional file 8: Table S8). Maximum number of genes were classified to be involved in metabolic process associated with spermatogenesis (180 genes), which have been reported to be associated with the active mark H3K4me3 in both human and mouse spermatid genome (33, 34). The most significant category was found to be belonging to cellular component organization with a p value of 2.7E-0.5. Interestingly, among these we found three important genes namely HMGB2, PYGO1 and TBPL1 which are known to be required for the spermatid nuclear differentiation. HMGB2 and TBPL1 are already identified as essential chromatin organizers that are necessary for proper chromocenter formation and/or maintenance, as revealed from targeted mutagenesis studies (35). In another study, loss of HMGB2 in mice affected H1T2 localization in late round spermatids and revealed intranuclear chromatin organization is critical for correct polar localization of H1T2 (29). Additionally we also found four other genes ASH1, CECR2, BRD2 and BRD4 belonging to bromodomain class of proteins, which are considered as the master regulators of maintenance of intact chromocenter in spermatids (Additional file 1: Table S1) (35).
Among the promoter centric H1T2 associated genes, we observed that 23 genes belonged to Transcription factor family upon GO: Molecular function analysis (Fig 4C; Additional file 9: Table S9). The identified occupancy peaks for these 23 transcription factors are shown in Figure 4D. As mentioned above, TATA box‐binding protein‐like 1 (TBPL1) is one among these, which has already been identified to be important for male sterility as evidenced by the sterile phenotype in Tbpl1−/− male mice due to a late and complete arrest of spermiogenesis at step 7 in stage VII seminiferous tubules (36). ING2, another candidate transcription factor whose promoter is bound to H1T2 is reported to be an essential regulator of mammalian spermatogenesis which is characterized by an increased expression level in testes and infertile knockout male mice. Furthermore a decreased ING2 expression level is also associated with defective spermatogenesis and male infertility in humans (37). E2F6, a part of the mammalian polycomb complex, is another transcription factor, expressed in the later stages of spermatogenesis, which has been identified as a master regulator through the coordinated action as both repressor and activator depending on the stage of expression. During male germ cell meiosis, E2F6 acts as an activator of meiosis-specific genes, demonstrated from moderately impaired spermatogenesis in E2F6 knockout mice (38). E2F6 recruits polycomb group proteins to function as a repressor of target genes during embryo development specifically associated to developmental patterning (39, 40). It is also essential for the long-term somatic silencing of certain male-germ-cell-specific genes and dispensable for cell-cycle regulation (41). H1T2 associates with the promoter of JMJD1C gene, which plays an indispensable role in mouse spermatogenesis as Jmjd1c-deficient males became infertile (42). It contributes to the long-term maintenance of the male germ line by promoting spermatogonial stem cell self-renewal by up-regulating Oct4 expression (43), required for mouse embryonic stem cell (ESC) self-renewal, mechanistically with the help of pluripotency factor KLF4 and maintains ESC identity and somatic cell reprogramming. Surprisingly, the promoter of KLF4, a pluripotency transcription factor which is highly expressed in male post meiotic germ cells is also bound to H1T2, which is however, dispensable for spermatogenesis. It is interesting that lack of KLF4 alone in male germ cells does not prevent spermiogenesis and male fertility (44). Deletion of Klf4 in male mouse germ cells has been shown to affect the post meiotic transcriptome including defined transcriptional regulators and identified as a key factor necessary for postnatal development and differentiation of the mouse testes (45). CAMTA1, one among the CAMTA gene family of transcription factors whose promoter is occupied by H1T2, is an important gene in both spermatogenesis and embryo development. CAMTA1 gene is important for various processes of fertility namely motility, capacitation, and acrosome reaction through calmodulin mediated calcium regulation. Additionally it is important for embryonic heart development, which is crucial for proper embryogenesis (46). The TSS region of CTCF, an architectural protein governing chromatin organization in sperm genome is also associated with linker histone H1T2, whose inactivation in male germ cells (Ctcf-cKO mice) resulted in impaired spermiogenesis and infertility (47). Interestingly, this study has also shown that H1T2 is one among those genes which are down-regulated in Ctcf-cKO mice and displays the same phenotypic features of H1T2 deleted condition namely abnormal head morphology, aberrant chromatin compaction, impaired protamine 1 incorporation (47). Being a highly conserved Zn finger protein, CTCF is critical for the three-dimensional organization of the genome and related functions during the development of various cell and tissue types, ranging from embryonic stem cells and gametes, to neural, muscle and cardiac cells (48)
Recent evidences accumulating in the literature are suggesting that sperm is programmed to support proper embryonic expression of genes encoding important embryonic regulators and in this context spermatids contribute epigenetic information required for proper embryonic gene expression (49). Our GO enrichment analysis also showed enrichment of H1T2 ChIP signals at the promoters of genes of several other developmental processes related pathways (Fig 4A; Additional file 8: Table S8): GO: 0044707~single-multicellular organism process (115 genes); GO: 0048856~anatomical structure development (105 genes); GO: 0044767~single-organism developmental process (106 genes); GO: 0050793~regulation of developmental process (43 genes). Some of the important candidate genes are TWIST2, TGLF1, CNOT3, SMARCC3 and ZFP whose functions in early embryonic differentiation process have been well documented (50-54).
Validation of H1T2 ChIP peaks of novel target genes by ChIP-PCR
We selected a representative set of 15 ChIP peaks for further validation of H1T2 occupancy and the IGV visualization of MACS ChIP peak and quantitative real time ChIP PCR assays of these peaks are shown in Fig 5A & B. The Primer pairs designed across the H1T2 peak regions are given in the supplementary file (Additional file 14: Table S14). IP signals were normalized relative to the signal obtained from the input chromatin and enrichment was calculated as percentage of the input. As can be seen, ChIP PCR assay clearly revealed an enrichment over IgG-negative control for all the ChIP samples analyzed (Fig5A). Among these, we have validated 7 representative transcription factor bound regions including TBPL1, ING2, JMJD2, HMGB2, SMARCC1, CTCF and E2F6 which confirmed the enrichment of H1T2 within the transcription start site regions of these regulatory genes which are either involved in spermatogenesis or embryogenesis (36, 55-58). H1T2 ChIP signals were also observed at loci of developmentally important genes including HOX (HOXB3, HOXB8, HOXC5); SOX (SOX12, SOX4, SOX14); FOX (FOXB1, FOXG1, FOXC1, FOXE1, FOXK2, FOXJ1, FOXM1); PAX (PAXX) and TBX (TBX1) gene clusters, where we observe at least one ChIP-seq peak located within 10 kb of the TSS (Additional file S1: Table S1). Representative genomic regions of the developmentally important loci, FOXB1, FOXC1 and SOX12 were also validated by ChIP PCR using H1T2 immunoprecipitated DNA as the template which confirmed significant enrichment of H1T2 in comparison to IgG immunoporecipiated chromatin control (Fig 5B). Earlier studies have also demonstrated the nucleosomal retention and bivalent histone marks (H3K4me3/H3K27me3) to be significantly enriched at these loci of developmental importance, including HOX, SOX, FOX, TBX, PAX, CDX, and GATA family transcription factors (33, 59). All together these results suggest a prominent role of the linker histone H1T2 in organizing specific spermatid chromatin domains to potentially regulate transcription of several developmentally important embryonic genes.
We also validated the occupancy of H1T2 with some representative examples of other classes of genomic loci like LINE elements, intronic, intergenic and CpG islands by ChIP PCR (Fig 5B). ChIP PCR analysis of the two representative loci of LINE repeat regions confirmed the association of H1T2 to the LINE (intergenic) regions which was the major fraction of genomic loci possessing H1T2 ChIP peaks, with significant enrichment in IP fractions in comparison to IgG pulldown fractions. Being the most successful retrotransposon, LINE elements presents an acute challenge to germ cells, which is managed by two repressive mechanisms of piRNA pathway and accumulation of repressive histone PTMs in addition to CpG methylation (31, 60, 61). LINE-1 (L1) subclass represents some 17–20% of the total human and mouse genomes, which seen to be still accumulating in the genome, as evidenced by the presence of L1 RNA and proteins in germ cells and infrequently in differentiated tissues (62, 63). Recently it has been observed that human L1 retrotransposition events likely occur in development rather than in the germline, using L1 RNAs transcribed in the embryo and in developing germ cells, suggesting active transcription of L1 in germ cells (64, 65). Additionally, representative loci of H1T2 bound regions like intron, intergenic and CpG were also validated using specific primers targeting the peak regions compared to a negative control locus devoid of any ChIP signal (Fig 5B).
Identification of proteins associated with H1T2 bound genomic regions by IP/MS analysis
To gain further insights into the H1T2 occupied genomic domains, we went ahead to characterize the functional protein complexes associated with H1T2 occupied genomic loci, by subjecting the chromatin IP samples to mass spectrometry analysis as detailed in the flow diagram in Figure 6A. Analysis of the proteome data identified 425 proteins with ≥ 4 unique peptides, which is usually considered to be significant (Additional file 10: Table S10). We found around 34 proteins with more than 15 unique number of peptides, which includes the proteins of cytoskeleton like actin, tubulin and intermediate filament proteins (plectin, desmin, lamin, vimentin), as listed in Figure 6B. Cytoskeletal dynamics in spermatogenesis is found to be regulated by the three networks (actin, tubulin and intermediate filament proteins) which function with each other to regulate different cellular processes, such as signaling, cell adhesion, cell motility, maintenance of cell polarity and protein targeting (66-70). The most significant H1T2-chromatin associated protein was myosin (Myh9) with 43 unique peptides, which is a reported component of acroplaxome which facilitates acrosomal spreading. Dynein (DYNC1H1) with 13 unique peptides (Additional file 10: Table S10) is also associated with H1T2-chromatin which is a component of manchette microtubules and spermatid nuclear envelope, pointing towards a potential function of H1T2 in nucleo-cytoskeleton interaction during spermiogenesis (14, 71, 72). Interestingly, H1T2 bound chromatin also interacts with lamin A/C protein encoded by LMNA gene, whose function is essential for cytoskeletal dynamics associated with spermatogenesis and its disruption leads to male infertility (73).
Functional annotation of the mass spectrometry identified proteins using DAVID program classified the proteins into the following groups: poly (A) RNA binding (133); ATP binding (48); structural constituent of ribosome (43); cadherin binding involved in cell-cell adhesion (31); mRNA binding (23); actin filament binding (17); ubiquitin protein ligase binding (22) respectively (Fig 6C; Additional file 11: Table S11). These groups have been summarized in Figure 6C. An important category of H1T2 bound chromatin interactome was ATP binding proteins (48 numbers), suggesting a potential role of H1T2 in regulating nuclear shaping involving the energy utilizing functional processes. Notably, ATP synthase subunits (ATP5B, ATP5F1, ATP5C1, ATP5O, ATP5A1, ATP5H) were also observed in this list, whose function is essential for germ cell maturation. Interestingly, their knockdown in Drosophila testes resulted in male infertility and abnormal spermatogenesis (74). We also carried out functional annotation of H1T2-chromatin interactome on the basis of biological process and the most significant categories are shown in the pie diagram (Fig 6C; Additional file 12: Table S12). There were 10 groups of proteins: metabolic process (303), cellular component organization or biogenesis (185), multi-organism process (75), detoxification (10), cellular process (356), reproductive process (52), localization (147), biological adhesion (50) and developmental process (141). Further, we also observed the association of the spermatid specific linker histone H1T2-bound chromatin with a large number of proteins involved in the sperm specific functions like sperm motility, spermatid differentiation and sperm-egg recognition (Fig 6D; Additional file 13: Table S13).
Male infertility has also been noted upon the ablation of several genes encoding RBPs (75). Indeed, RNA binding proteins were one of the major classes of proteins associated to H1T2 bound chromatin in spermatids (Fig 6C; Additional file 11: Table S11). The major function attributed to spermatid specific RBPs is the post‐transcriptional regulation of mRNAs which are highly expressed in germ cells (75). Specifically, the mRNPs functions in the storage of silenced transcripts until factors in the elongation phase of spermiogenesis trigger their translation to support final steps in spermatozoan development and fertilization (76-78). The mRNA binding proteins interacting with H1T2 bound chromatin are listed in Figure 6D. Numerous RBPs are synthesized solely in late phases of spermiogenesis, ensuring a temporal regulation of their target mRNAs (79). Embryonic Lethal Abnormal Vision (ELAV) L1 is one such candidate RBP pulled down by H1T2 antibody which is reported as a key regulator of posttranscriptional regulation in spermatocytes, whose targeted deletion results in male sterility phenotype (80). More recent studies have supported the mRNA regulatory role of Elavl1 by specifically associating to Tnp mRNAs to control the delayed timing of their translation (81). H1T2-chromatin IP/MS analysis also identified YbX2, another potential mRNA repressor involved in the regulation of translation whose knockout resulted in male infertility and azoospermia condition (82). Notably we also found Ddx5 in our list of H1T2-chromatin interacting proteins, which is considered to be an essential protein for both transcriptional and posttranscriptional roles in the maintenance and function of spermatogonia (83). PABP1 was the other important protein pulled down with H1T2-chromatin, whose function has been extensively characterized in germ cells and shown to have important roles in mRNA stability and translation (84). Other candidate RBPs includes PSPC1, PTBP1, PTBP2, SFPQ, RBM14, SNRPs and HNRNPs. We went ahead to further validate the association of a representative set of proteins with H1T2 bound chromatin by western blot analysis (Fig 6E). More recently, LINE repeats have been reported to harbor repressive RBPs thereby contributing to the evolution of new, lineage-specific transcripts in mammals (85). In this context, we did observe a substantial overlap between the H1T2-chromatin interacting proteins and LINE1 ORF1 interactome which function in the regulation of retrotransposons (85, 86). It is worth noting here that LINE Rn_L1 subclass of LINE elements that we have observed to be associated with H1T2, is indeed an active element containing ORF1 and ORF2 within the repeat (32).
Identification of PTMs of histones associated with H1T2 occupied chromatin domain
To investigate the epigenetic status of H1T2 associated chromatin in terms of histone PTMs, acid soluble fraction was prepared from H1T2 immunoprecipitated chromatin and subjected to mass spectrometry analysis. Histone PTMs associated to these H1T2 bound chromatin loci were identified to be acetylation marks like H2BK21ac, H2AK95ac, H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K18ac, H3K23ac and H3K27ac and methylation marks like H3K27me2 and H3K79me2 (Fig 7A). Western blot analysis confirmed a combination of acetylated H4 peptides (H4K5ac, H4K12ac, and H4K16ac) which are activation marks to be highly enriched in the immunoprecipitated chromatin, whereas no signals were observed in IgG control samples (Fig 7B). Recent evidences have shown the involvement of H1T2 in the initiation of chromatin remodeling in spermatogenesis wherein H4 acetylation acts as a key to open up the chromatin in the doughnut structure in the apical region to facilitate the histone replacement in round spermatids (7). Another study has also revealed the enrichment of BRD4, H3K9ac, and H4K5ac, H4K8ac, H4K12ac, and H4K16ac towards TSSs of active genes in round spermatids, and their enrichment correlates with transcription levels (87). Further analysis also revealed the enrichment of other active histone PTMs like H3K4me3, H3K9ac, H3K27ac, H3K4me1 and H3K79me2, where as the repressive modifications like H4K20me3, H3K9me3, H3K27me3 were absent in the H1T2 immunoprecipitated chromatin fraction (Fig 7B). Incorporation of H1T2 in early round spermatids can be considered as an additional player in the chromatin reorganization process in addition to H3K79 methylation accompanying H4 acetylation as has been reported previously (88, 89). Notably, we also found H1T2 occupied chromatin domains to harbor the histone variants like H3.3 and TH2B as demonstrated by IP/western blot. These core histone variants have been shown to be involved in the formation of transitional subnucleosomal structures during histone to transition proteins/protamine transition (90-93). Taken together, the results presented so far suggests a unique property to the spermatid specific H1, H1T2 to organize the polar domain of the spermatid genome by clustering the actively transcribed or to be transcribed genomic domains and in association with extensive number of chromatin interacting partners and cytoskeleton mediated mechanical force. H1T2 may therefore contribute to the chromatin remodeling process as well as nuclear shaping event that occurs during the elongation of spermatid nucleus in rodents.