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Fig. 4 | Epigenetics & Chromatin

Fig. 4

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

Fig. 4

HILS1 primarily associates with intergenic and intronic regions. ChIP sequencing was done using antibody against HILS1 in rat elongating/condensing spermatids. a Dot blot analysis of affinity purified antibody against HILS1 with respective peptide. b Western blot analysis of the acid extracts from rat liver (lane 1) and testis (lane 2) with antibody against HILS1. c Western blot analysis of acid-soluble proteins from 25-day postnatal (lane 1) and 50-day postnatal (lane 2) rat testis nuclei with anti-HILS1 antibodies in the presence or absence of peptide competition with a 200-fold molar excess of the peptide used for antibody generation, as indicated. Western blot analysis with anti-TP2 and anti-H3 antibodies served as a positive control. d Western blot analysis of acid extracts of epididymal sperm using HILS1 antibody. e Rat testis nuclei were separated into soluble (nucleoplasm; lane 1) and insoluble (chromatin; lane 2) fractions. Western blot analysis was done using antibody against HILS1. Anti-GRP78 antibodies and anti-histone H4 antibodies served as a positive control to probe the soluble fraction (lane 1) and insoluble fraction (lane 2), respectively. f Western blot analysis of the pull-down fractions with α HILS1. Input (lane 1), anti-HILS1 antibody (lane 2), anti-HILS1 antibodies + peptide (lane 3), IgG (lane 4). No signal was observed with pre-immune IgG, and heavy and light chains are indicated by asterisk. Antibody specificity is analyzed by confirming the pulled-down bands as HILS1 by mass spectrometry (Additional file 1: Figure S1). g Box plot showing the log2fold enrichment of HILS1 peaks (y-axis) across different rat chromosomes. h Box plot representing the HILS1 associated average peak length (y-axis) across different chromosomes (x-axis) indicating that HILS1 peaks are broad in nature. i Distribution of log2fold change of the HILS1 aligned reads over the input control that fall within ± 1000 bp of the known 17057 CpG islands in Rattus norvegicus genome rn5. j Pie chart distribution of HILS1 peaks for various genomic features in rat spermatids. Most of the peaks are bound to intergenic and intronic regions. k Bar diagram of number of occurrences of peaks across repeat elements. l Bar diagram showing the number of occurrences of peaks across different subclasses of LINE-1 repeat elements

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