Polycomb protein SCML2 associates with USP7 and counteracts histone H2A ubiquitination in the XY chromatin during male meiosis

Abstract

Polycomb group proteins mediate transcriptional silencing in diverse developmental processes. Sex chromosomes undergo chromosome-wide transcription silencing during male meiosis. Here we report that mouse SCML2 (Sex comb on midleg-like 2), an X chromosome-encoded polycomb protein, is specifically expressed in germ cells, including spermatogonia, spermatocytes, and round spermatids. SCML2 associates with phosphorylated H2AX and localizes to the XY body in spermatocytes. Loss of SCML2 in mice causes defective spermatogenesis, resulting in sharply reduced sperm production. SCML2 interacts with and recruits a deubiquitinase, USP7, to the XY body in spermatocytes. In the absence of SCML2, USP7 fails to accumulate on the XY body, whereas H2A monoubiquitination is dramatically augmented in the XY chromatin. Our results demonstrate that the SCML2/USP7 complex constitutes a novel molecular pathway in modulating the epigenetic state of sex chromosomes during male meiosis.

Figure 1. Expression and localization of SCML2 during mouse spermatogenesis.

(A) Domain structure of mouse SCML2 protein (XP_006528733.1). MBT, malignant brain tumor repeat; DUF3588, also called Scm-like embedded domain (SLED) [11]; SAM, sterile alpha motif. (B) Western blot analysis of SCML2 in adult mouse tissues. ACTB serves as a control. (C) Western blot analysis of SCML2 on cytoplasmic extract, nuclear extract, and chromatin from postnatal day 20 testis. SYCP2, a synaptonemal complex protein, serves as a control [44]. (D) Expression and localization of SCML2 in male germ cells. Testis sections from 2-month-old mice were immunostained with SCML2 and γH2AX antibodies. Nuclei were stained with DAPI. The inset is an enlarged view of a pachytene spermatocyte with the XY body indicated. Abbreviations: Sertoli, Sertoli cells; Spg, spermatogonium; Pa, pachytene spermatocyte; RS, round spermatid; ES, elongated spermatid. Scale bar, 50 μm. (E) Localization of SCML2 to the XY body in wild type spermatocytes. Spread nuclei were immunostained with anti-SCML2 and anti-SYCP3 antibodies. SCML2 staining alone are shown in bottom panels. XY chromosomes are shown in insets. (F) Co-immunoprecipitation of SCML2 and γH2AX from wild type (Scml2 ^+/Y) testicular nuclear extracts. Scml2 ^-/Y testis was used as a negative control. (G) Lack of preferential accumulation of SCML2 in the pseudo sex body in Spo11 ^-/- zygotene-like spermatocytes.

Figure 2. Defective spermatogenesis in Scml2 ^-/Y mice.

(A) Western blot analysis of wild type and Scml2 ^-/Y testes. (B) Dramatic testis weight reduction (per pair of testes) in Scml2 ^-/Y mice. (C) Sharp reduction in sperm count (per pair of cauda epididymides) in Scml2 ^-/Y mice. The body weight of age-matched male mice was similar. Body weight at the 2-month age: wild type, 28.0 ± 1.5 g and Scml2 ^-/Y, 28.6 ± 1.0 g (n = 3 per genotype). Body weight at the 8-month age: wild type, 39.4 ± 4.7 g and Scml2 ^-/Y, 39.3 ± 5.5 g (n = 3 per genotype). (D) Histology of a 4-month-old wild-type testis at low magnification. (E-I) Variable spermatogenic defects in tubules from 4-month-old Scml2 ^-/Y testes. Testis section is shown in low magnification (E). Spermatogenesis in some tubules (F) is relatively normal. Apparently apoptotic spermatocytes are indicated by arrowheads (G). A layer of lost spermatocytes is marked by a dashed line (H). Some tubules (I) exhibit nearly complete depletion of pachytene spermatocytes. Vacuoles are indicated by arrows (I). Abbreviations (F-I): Pa, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. Scale bars, 50 μm.

Figure 3. Apoptosis of spermatocytes in adult Scml2 ^-/Y testes.

Based on the expression of histone H1t in pachytene spermatocytes, seminiferous tubules were divided into two groups: H1t-negative (early pachytene stage) and H1t-positive (mid-late pachytene stage). The number of TUNEL-positive cells per tubule cross-section was counted. (A) Apoptosis in both H1t-negative and H1t-positive Scml2 ^-/Y seminiferous tubules. Apoptotic cells presumably corresponded to pachytene spermatocytes. Pa: pachytene spermatocytes. Scale bars, 25 μm. (B) Count of apoptotic cells. 100 tubules from adult Scml2 ^-/Y testes and 239 tubules from wild type testes were analyzed.

Figure 4. SCML2 associates with USP7 in the XY body.

(A) Identification of SCML2-associated proteins from 20-day testes by immunoprecipitation (IP) and mass spectrometry. The extra bands (vertical line) in the wild type IP and the corresponding region (vertical line) in the Scml2 ^-/Y IP were subjected to mass spectrometry. (B) Co-immunoprecipitation of SCML2 with USP7 in testes. Nuclear protein extracts prepared from 20-day testes were used. Scml2 ^-/Y testes were used as a negative control. Note that the USP7 antibody (Bethyl Laboratory) recognizes two bands with the lower band being more abundant. Both USP7 bands were co-immunoprecipitated with SCML2. However, the nature of the two USP7 isoforms is unknown. (C) Immunolocalization of USP7 and γH2AX in wild type and Scml2 ^-/Y seminiferous tubules from 8-week-old mice. Testicular frozen sections were used for double immunostaining. Scale bars, 25 μm. (D) Localization of USP7 to the XY body in wild type spermatocytes. Spread nuclei from prophase I spermatocytes (from zygotene to diplotene stages) were immunostained with anti-USP7 and anti-SYCP3 antibodies. USP7 localization alone is shown in bottom panels.

Figure 5. Increased H2A monoubiquitination in the XY body in Scml2-deficeint pachytene and diplotene spermatocytes.

Distribution of monoubiquitinated H2A (uH2A) in wild type (A) and Scml2 ^-/Y (B) prophase I spermatocytes were examined by immunostaining of spread nuclei with anti-SYCP3 (red) and anti-uH2A (green) antibodies. uH2A staining alone is shown in the bottom panels. The XY bodies are indicated by arrowheads and shown in the insets. Notably, uH2A localizes to and radiates from the synaptonemal complexes in wild type and Scml2 ^-/Y pachytene spermatocytes.