Genome-wide occupancy reveals the localization of H1T2 (H1fnt) to repeat regions and a subset of transcriptionally active chromatin domains in rat spermatids.

BACKGROUND: H1T2/H1FNT is a germ cell-specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone-to-protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown. RESULTS: Sequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome-wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions (promoter-TSS, exons and introns) of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2-associated chromatin mainly as nucleo-skeleton components, RNA-binding proteins and chaperones. CONCLUSIONS: Linker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids.

Spermatid-specific linker histone HILS1 is a poor condenser of DNA and chromatin and preferentially associates with LINE-1 elements.

BACKGROUND: Linker histones establish and maintain higher-order chromatin structure. Eleven linker histone subtypes have been reported in mammals. HILS1 is a spermatid-specific linker histone, and its expression overlaps with the histone-protamine exchange process during mammalian spermiogenesis. However, the role of HILS1 in spermatid chromatin remodeling is largely unknown. RESULTS: In this study, we demonstrate using circular dichroism spectroscopy that HILS1 is a poor condenser of DNA and chromatin compared to somatic linker histone H1d. Genome-wide occupancy study in elongating/condensing spermatids revealed the preferential binding of HILS1 to the LINE-1 (L1) elements within the intergenic and intronic regions of rat spermatid genome. We observed specific enrichment of the histone PTMs like H3K9me3, H4K20me3 and H4 acetylation marks (H4K5ac and H4K12ac) in the HILS1-bound chromatin complex, whereas H3K4me3 and H3K27me3 marks were absent. CONCLUSIONS: HILS1 possesses significantly lower α-helicity compared to other linker histones such as H1t and H1d. Interestingly, in contrast to the somatic histone variant H1d, HILS1 is a poor condenser of chromatin which demonstrate the idea that this particular linker histone variant may have distinct role in histone to protamine replacement. Based on HILS1 ChIP-seq analysis of elongating/condensing spermatids, we speculate that HILS1 may provide a platform for the structural transitions and forms the higher-order chromatin structures encompassing LINE-1 elements during spermiogenesis.

Isolation, characterization and quantification of tricin and flavonolignans in the medicinal rice Njavara (Oryza sativa L.), as compared to staple varieties.

Njavara is an important medicinal rice variety of Kerala, India, widely used in Ayurveda as a 'health food' and in the treatment of rheumatoid arthritis, paralysis, neurodegenerative diseases and in rejuvenation therapy. Phytochemical investigations and spectroscopic studies of the diethyl ether fraction of methanolic extract of Njavara Black (NB) rice bran gave three important compounds namely, tricin and two rare flavonolignans- tricin 4'-O-(erythro-ß-guaiacylglyceryl) ether and tricin 4'-O-(threo-ß-guaiacylglyceryl) ether. The EC(50) values of these compounds in DPPH system were 90.39, 352.04 and 208.1 µg/ml, respectively. Quantification of the compounds by HPLC in NB and staple, non-medicinal rice varieties Sujatha (SJ) and Palakkadan Matta (PM) showed that tricin is present 39.64 and 16.12 fold higher in NB, compared to SJ and PM, respectively. This is the first report on the occurrence of tricin at significantly higher levels in Njavara and occurrence of the two flavonolignans in Oryza sativa species. Of the three compounds, tricin and the threo- form of flavonolignan showed anti-inflammatory effect of >65% after 5 h, at 2 mg/kg, in carrageenan-induced, paw edema experiments in rats. The results of the study corroborate with the preferential use of Njavara in indigenous medicine, over staple varieties.