Testis-specific Linker Histone H1t Is Multiply Phosphorylated during Spermatogenesis

Abstract

During normal spermatogenesis, the testis-specific linker histone H1t appears at pachytene stage becomes phosphorylated in early spermatids and disappears in late spermatids. Using reversed-phase and hydrophilic interaction liquid chromatography, H1t from rat and mouse testes was isolated, subjected to enzymatic digestion, and analyzed by mass spectrometry. We observed different phosphorylated states of H1t (mono-, di-, and triphosphorylated) as well as the unphosphorylated protein.Tandem mass spectrometry and immobilized metal ion affinity chromatography experiments with MS/MS/MS and multistage activation were utilized to identify five phosphorylation sites on H1t from rats. Phosphorylation occurs on both serine and threonine residues, whereas only two of these sites were located on peptides containing the CDK consensus motif (S/T)PXZ. Rat H1t phosphorylation starts first by phosphorylation of the nonconsensus motif SPKS in the COOH-terminal domain, namely at Ser-140 and to a smaller degree at a further nonconsensus motif at Ser-186. This is followed by phosphorylation of Ser-177 and Thr-155, both located in CDK consensus motifs. A single phosphorylation site at Ser-8 in the NH 2 -terminal tail was also found. Mouse H1t lacks Ser-186 and is phosphorylated at up to four sites. In contrast to somatic linker histones, no strict order of increasing phosphorylation could be detected in H1t. Thus, it appears that not the order of up-phosphorylation but the number of the phosphate groups is necessary for regulated chromatin decondensation, thus facilitating the substitution of H1t by transition proteins and protamines.Mammalian spermatogenesis is a complex, strictly organized process, including proliferation and differentiation. After several mitotic divisions of spermatogonia, germ cells differentiate into spermatocytes and pass through two consecutive meiotic divisions, resulting in haploid round cells termed spermatids. These spermatids then evolve into highly condensed and transcriptionally inert sperm through a process known as spermiogenesis (for a review, see Ref.

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

“…Most of the sites could be identified with both CE and LC, but some were found with one method only. We confirmed all five of the phosphorylation sites that we recently reported for rat H1t (14) and identified three additional, novel phosphorylation sites in this subtype. Two of these sites could be assigned unambiguously to Ser-40 and Ser-79.…”

“…Over the past several years, considerable efforts have been expended to develop methods to identify the specific sites of histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the dominant technique for their characterization (7)(8)(9)(10)(11)(12)(13)(14). However, because histone proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still problematic, as digestion with many commonly used enzymes (e.g.…”

Site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different processes during the cell cycle

“…Further heterogeneity in the H1 histone arises from various posttranslational modifications at several positions including phosphorylation. Phosphorylation occurs at specific serine and threonine residues in the N-and C-terminal tail regions of histone H1 (Sarg et al 2006(Sarg et al , 2009Talasz et al 1996). Histone H1 phosphorylation is cell cycle-dependent with low phosphorylation in G1 and increasing rate and extent throughout S-and G2-phase reaching a maximum during mitosis (Talasz et al 1996).…”

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 2: Changes in spermatid organelles associated with development of spermatozoa

“…H1t binds to linker DNA and nucleosome core particles and appears to facilitate the folding of chromatin into a more compact structure. Multiple sites of the H1t are phosphorylated and these phosphate groups are needed for regulating chromatin decondensation and thus facilitating the substitution of H1t by transition proteins and protamines (Sarg et al, 2009). The testis-specific linker histone H1t gene is expressed in primary spermatocytes, and its regulation whether activated (regulatory factor RFX2 [ID: T2-354]) or repressed (DNA methylation) is discussed in reviews by Grimes et al (2003) and Grimes (2004).…”