HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis

Abstract

Chromatin remodeling is a major event that occurs during mammalian spermiogenesis, the process of spermatid maturation into spermatozoa. Nuclear condensation during spermiogenesis is accomplished by replacing somatic histones (linker histone H1 and core histones) and the testis-specific linker histone, H1t, with transition proteins and protamines. It has long been thought that H1t is the only testis-specific linker histone, and that all linker histones are replaced by transition proteins, and subsequently by protamines during spermiogenesis. Here, we report the identification and characterization of a spermatid-specific linker histone H1-like protein (termed HILS1) in the mouse and human. Both mouse and human HILS1 genes are located in intron 8 of the alpha-sarcoglycan genes. HILS1 is highly expressed in nuclei of elongating and elongated spermatids (steps 9-15). HILS1 displays several biochemical properties that are similar to those of linker histones, including the abilities to bind reconstituted mononucleosomes, produce a chromatosome stop during micrococcal nuclease digestion, and aggregate chromatin. Because HILS1 is expressed in late spermatids that do not contain core histones, HILS1 may participate in spermatid nuclear condensation through a mechanism distinct from that of linker histones. Because HILS1 also belongs to the large winged helix/forkhead protein superfamily, HILS1 may also regulate gene transcription, DNA repair, and/or other chromosome processes during mammalian spermiogenesis.

Fig. 1.

Analysis of Hils1 mRNA in mouse and human tissues. (A) Northern blot analysis of Hils1 mRNA expression in mouse tissues. Total RNA (15 g) from heart (He), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), brain (Br), stomach (St), intestine (In), testis (Te), ovary (Ov), and uterus (Ut) was electrophoresed, transferred, and probed with Hils1 or 18S rRNA cDNAs. (B) RT-PCR analysis of Hils1 and Hprt mRNA in multiple mouse tissues. (C) RT-PCR analysis of HILS1 and ACTIN mRNA in human tissues. The abbreviations are similar to A and include colon (Co), prostate (Pr), and thymus (Th).

Fig. 2.

HILS1 genomic and protein structures. (A) Genomic organization and domain structures of the mouse and human HILS1 proteins. The mouse Hils1 is located in intron 8 of the mouse α-sarcoglycan gene (SGCA), and a similar genomic arrangement is conserved in humans. Arrows represent the transcriptional orientations of the genes. Each protein contains a globular domain (GD), which is flanked by an N-terminal domain (ND) and a C-terminal domain (CD). The conserved putative phosphorylation sites are marked. (B) Alignment analysis of the globular domains of histone H1 family members. Identical residues are highlighted, and residues conserved in at least 8 of 10 members are marked with asterisks. Amino acid identity between the globular domains of the mouse HILS1 and other family members is shown. (C) Phylogenetic tree of linker histone H1 family proteins. (D) Predicted 3D structure of mouse HILS1 versus histone H1a. Cylinders represent α-helical bundles, and arrows represent β-sheets. The angles of rotation are shown.

Fig. 3.

Mouse HILS1 protein expression and localization. (A) Western blot analysis of HILS1 in mouse tissues. (B) HILS1 expression in developing testes. Proteins isolated from testes at different postnatal days (d) were analyzed by Western blot. (C) Stage-specific expression of HILS1 protein in the adult mouse testis. Seminiferous tubule segments as shown were dissected, and protein was prepared. ACTIN was used as a loading control (A-C). (D) Immunohistochemical detection of HILS1. Stages of adult mouse seminiferous tubules are marked with Roman numerals, and brown diaminobenzidine staining represents positive signals. HILS1 immunoreactivity is confined to step 9-15 spermatids. (E) Schematic summary of the expression profiles of HILS1, H1t, TNP1, TNP2, PRM1, and PRM 2 during spermiogenesis based on Figs. 3 and 9, and previously reported data on H1t expression (11, 12). Arabic numbers represent the steps of spermatids, and Roman numerals indicate the tubule stages. Bars represent the expression windows of these NPs in spermatids.

Fig. 4.

Biochemical characteristics of HILS1 in vitro.(A) Analysis of binding activity of histone H1a and HILS1 to mononucleosomes by gel shift assay. Nucleosomes reconstituted by using core histone particles prepared from mouse liver and digoxigenin-11-ddUTP end-labeled 210-bp mouse mammary tumor virus (MMTV) LTR DNA were incubated with 0, 0.375, 0.75, 1.5, 3, 6, and 12 ng of H1a or HILS1, corresponding to 0, 1, 2, 4, 8, 16, and 32 nM (lanes 2-8, respectively). Lane 1, free 210-bp DNA. Positions of free DNA, nucleosome (Nuc), and histone H1-nucleosome complexes (Nuc-H1) are indicated. (B) Chromatin aggregation properties of HILS1. Polynucleosomes reconstituted with digoxigenin-11-ddUTP end-labeled 1.3-kb MMTV LTR DNA were incubated with 0, 12, 24, 36, 48, 72, and 96 ng of HILS1, corresponding to 0, 45, 90, 135, 180, 270, and 360 nM (lanes 2-8, respectively). Lane 1, free 1.3-kb DNA. (C) Chromatosome stop assay. Nucleosomes reconstituted with digoxigenin-11-dUTP-labeled MMTV LTR promoter DNA were incubated with HILS1 [HILS1:DNA ratio (wt/wt) of 0.4 (lanes 2-4), and 0.1 (lanes 5-7)] and digested with 0.15, 0.3, or 0.6 units of micrococcal nuclease (MCN) for 6 min at 37°C. DNA fragments corresponding to nucleosome core particle (146 bp) and chromatosome (168 bp and arrow) are indicated. (D) Gel shift assay of binding activity of HILS1 and H1a to naked DNA. Digoxigenin-11-ddUTP end-labeled 210-bp MMTV LTR DNA was incubated with 0.375, 0.75, 1.5, 3, 6, and 12 ng of H1a and HILS1, corresponding to 1, 2, 4, 8, 16, and 32 nM (lanes 1-6, respectively).

Fig. 5.

Detection of HILS1 in total nuclear and chromatin protein preparations. (A) Coomassie blue-stained acid-urea 15% polyacrylamide gel (a) and Western blot analyses of HILS1 (d), H1 (b), and TNP1 (c). Total BNPs from unfractionated mouse testis nuclei were used as a marker (a). (B) Quantitative analysis of HILS1 in total CPs and total NPs. Ten grams of total NP or CP was fractionated through a 4-12% polyacrylamide gel. Serial dilution of recombinant mouse HILS1 protein was prepared and loaded in each lane as shown. After chemiluminescent detection, the film was scanned and quantified.