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Identification of a variant-specific phosphorylation of TH2A during spermiogenesis

View Article: PubMed Central - PubMed

ABSTRACT

Tissue-specific histone variant incorporation into chromatin plays dynamic and important roles in tissue development. Testis is one such tissue, and a number of testis-specific histone variants are expressed that have unique roles. While it is expected that such variants acquire post-transcriptional modifications to be functional, identification of variant-specific histone modifications is challenging because of the high similarity of amino acid sequences between canonical and variant versions. Here we identified a novel phosphorylation on TH2A, a germ cell-specific histone H2A variant. TH2A-Thr127 is unique to the variant and phosphorylated concomitant with chromatin condensation including spermiogenesis and early embryonic mitosis. In sperm chromatin, phosphorylated TH2A-Thr127 (=pTH2A) is co-localized with H3.3 at transcriptional starting sites of the genome, and subsequently becomes absent from the paternal genome upon fertilization. Notably, pTH2A is recurrent and accumulated in the pericentromeric heterochromatin of both paternal and maternal chromosomes in the first mitosis of embryos, suggesting its unique regulation during spermiogenesis and early embryogenesis.

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Identification of pTH2A in mouse spermatozoa.(a) An experimental scheme to prepare nuclear fractions from spermatozoa. Proteins are biochemically divided into three fractions (S1, S2, and Ppt). Silver staining and western blot analyses of each fraction are as indicated. The red box indicates the area to be digested and subjected to LC-MS analysis. (b) Representative MS/MS spectrum of pTH2A peptides (upper panel). The y-axis indicates intensity of MS/MS spectra, and the x-axis indicates m/z. Phosphorylated threonine is highlighted in red, and the identification number for each peptide is summarized (lower panel). (c) Alignment of amino acid sequences between mouse H2A, mouse TH2A, and human TH2A. Black arrow, start of the C-terminal tails; red box, the phosphorylated threonine in mouse TH2A; green line, epitope sequence for generation of the anti-pTH2A antibody; Roman numeral, number of amino acid from the N-terminus. (d) Western blot analysis of sperm S2 fraction treated or untreated with lambda protein phosphatase (λPPase). Experimental procedure (left panel) and representative images of western blot analysis (right panel) are indicated. (e) Peptide blocking assay. Indicated peptides were added to the diluted primary antibody during the reaction. (f) Western blot analysis of pTH2A (upper panel) and TH2A (lower panel). Protein extracts of HEK293T cells overexpressing FLAG-tagged H2A (F-H2A) and -TH2A (F-TH2A), whole testicular cells, germ stem cells (GS cells), and spermatozoa were analyzed. Number of biological replicates of each experiment is shown as n.
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f1: Identification of pTH2A in mouse spermatozoa.(a) An experimental scheme to prepare nuclear fractions from spermatozoa. Proteins are biochemically divided into three fractions (S1, S2, and Ppt). Silver staining and western blot analyses of each fraction are as indicated. The red box indicates the area to be digested and subjected to LC-MS analysis. (b) Representative MS/MS spectrum of pTH2A peptides (upper panel). The y-axis indicates intensity of MS/MS spectra, and the x-axis indicates m/z. Phosphorylated threonine is highlighted in red, and the identification number for each peptide is summarized (lower panel). (c) Alignment of amino acid sequences between mouse H2A, mouse TH2A, and human TH2A. Black arrow, start of the C-terminal tails; red box, the phosphorylated threonine in mouse TH2A; green line, epitope sequence for generation of the anti-pTH2A antibody; Roman numeral, number of amino acid from the N-terminus. (d) Western blot analysis of sperm S2 fraction treated or untreated with lambda protein phosphatase (λPPase). Experimental procedure (left panel) and representative images of western blot analysis (right panel) are indicated. (e) Peptide blocking assay. Indicated peptides were added to the diluted primary antibody during the reaction. (f) Western blot analysis of pTH2A (upper panel) and TH2A (lower panel). Protein extracts of HEK293T cells overexpressing FLAG-tagged H2A (F-H2A) and -TH2A (F-TH2A), whole testicular cells, germ stem cells (GS cells), and spermatozoa were analyzed. Number of biological replicates of each experiment is shown as n.

Mentions: To identify novel histone modifications in mouse spermatozoa, we isolated caudal epididymal sperm and extracted the chromatin via a combination of dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and micrococcus nuclease (MNase) treatment (Figs 1a and S5a). The solubilized fraction (S2) was resolved by SDS-PAGE (Fig. 1a), and proteins around 10–20 kilodalton, where core histones should be enriched, were subjected to liquid chromatography-mass spectrometry (LC-MS). This analysis revealed acetylation on the histones H4-K5, -K8, -K12 and -K16, and H4-K20 mono-methylation (Fig. S1b,c), which are consistent with previous reports28. Interestingly, a phosphorylation site at Thr127 of TH2A (TH2A-pT127 or pTH2A), which is located at the end of C-terminus of TH2A and not conserved in canonical H2A (Fig. 1b,c), was also identified. Although the C-terminal sequence of human TH2A is not identical to that of mice, the amino acid corresponding to mouse Thr127 is Ser129 in human and surrounding sequences are well-conserved (Fig. 1c).


Identification of a variant-specific phosphorylation of TH2A during spermiogenesis
Identification of pTH2A in mouse spermatozoa.(a) An experimental scheme to prepare nuclear fractions from spermatozoa. Proteins are biochemically divided into three fractions (S1, S2, and Ppt). Silver staining and western blot analyses of each fraction are as indicated. The red box indicates the area to be digested and subjected to LC-MS analysis. (b) Representative MS/MS spectrum of pTH2A peptides (upper panel). The y-axis indicates intensity of MS/MS spectra, and the x-axis indicates m/z. Phosphorylated threonine is highlighted in red, and the identification number for each peptide is summarized (lower panel). (c) Alignment of amino acid sequences between mouse H2A, mouse TH2A, and human TH2A. Black arrow, start of the C-terminal tails; red box, the phosphorylated threonine in mouse TH2A; green line, epitope sequence for generation of the anti-pTH2A antibody; Roman numeral, number of amino acid from the N-terminus. (d) Western blot analysis of sperm S2 fraction treated or untreated with lambda protein phosphatase (λPPase). Experimental procedure (left panel) and representative images of western blot analysis (right panel) are indicated. (e) Peptide blocking assay. Indicated peptides were added to the diluted primary antibody during the reaction. (f) Western blot analysis of pTH2A (upper panel) and TH2A (lower panel). Protein extracts of HEK293T cells overexpressing FLAG-tagged H2A (F-H2A) and -TH2A (F-TH2A), whole testicular cells, germ stem cells (GS cells), and spermatozoa were analyzed. Number of biological replicates of each experiment is shown as n.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384234&req=5

f1: Identification of pTH2A in mouse spermatozoa.(a) An experimental scheme to prepare nuclear fractions from spermatozoa. Proteins are biochemically divided into three fractions (S1, S2, and Ppt). Silver staining and western blot analyses of each fraction are as indicated. The red box indicates the area to be digested and subjected to LC-MS analysis. (b) Representative MS/MS spectrum of pTH2A peptides (upper panel). The y-axis indicates intensity of MS/MS spectra, and the x-axis indicates m/z. Phosphorylated threonine is highlighted in red, and the identification number for each peptide is summarized (lower panel). (c) Alignment of amino acid sequences between mouse H2A, mouse TH2A, and human TH2A. Black arrow, start of the C-terminal tails; red box, the phosphorylated threonine in mouse TH2A; green line, epitope sequence for generation of the anti-pTH2A antibody; Roman numeral, number of amino acid from the N-terminus. (d) Western blot analysis of sperm S2 fraction treated or untreated with lambda protein phosphatase (λPPase). Experimental procedure (left panel) and representative images of western blot analysis (right panel) are indicated. (e) Peptide blocking assay. Indicated peptides were added to the diluted primary antibody during the reaction. (f) Western blot analysis of pTH2A (upper panel) and TH2A (lower panel). Protein extracts of HEK293T cells overexpressing FLAG-tagged H2A (F-H2A) and -TH2A (F-TH2A), whole testicular cells, germ stem cells (GS cells), and spermatozoa were analyzed. Number of biological replicates of each experiment is shown as n.
Mentions: To identify novel histone modifications in mouse spermatozoa, we isolated caudal epididymal sperm and extracted the chromatin via a combination of dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and micrococcus nuclease (MNase) treatment (Figs 1a and S5a). The solubilized fraction (S2) was resolved by SDS-PAGE (Fig. 1a), and proteins around 10–20 kilodalton, where core histones should be enriched, were subjected to liquid chromatography-mass spectrometry (LC-MS). This analysis revealed acetylation on the histones H4-K5, -K8, -K12 and -K16, and H4-K20 mono-methylation (Fig. S1b,c), which are consistent with previous reports28. Interestingly, a phosphorylation site at Thr127 of TH2A (TH2A-pT127 or pTH2A), which is located at the end of C-terminus of TH2A and not conserved in canonical H2A (Fig. 1b,c), was also identified. Although the C-terminal sequence of human TH2A is not identical to that of mice, the amino acid corresponding to mouse Thr127 is Ser129 in human and surrounding sequences are well-conserved (Fig. 1c).

View Article: PubMed Central - PubMed

ABSTRACT

Tissue-specific histone variant incorporation into chromatin plays dynamic and important roles in tissue development. Testis is one such tissue, and a number of testis-specific histone variants are expressed that have unique roles. While it is expected that such variants acquire post-transcriptional modifications to be functional, identification of variant-specific histone modifications is challenging because of the high similarity of amino acid sequences between canonical and variant versions. Here we identified a novel phosphorylation on TH2A, a germ cell-specific histone H2A variant. TH2A-Thr127 is unique to the variant and phosphorylated concomitant with chromatin condensation including spermiogenesis and early embryonic mitosis. In sperm chromatin, phosphorylated TH2A-Thr127 (=pTH2A) is co-localized with H3.3 at transcriptional starting sites of the genome, and subsequently becomes absent from the paternal genome upon fertilization. Notably, pTH2A is recurrent and accumulated in the pericentromeric heterochromatin of both paternal and maternal chromosomes in the first mitosis of embryos, suggesting its unique regulation during spermiogenesis and early embryogenesis.

No MeSH data available.


Related in: MedlinePlus