ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline

Abstract

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

Keywords: ATF7IP2/MCAF2; H3K9me3; constitutive heterochromatin; gene activation; meiosis; meiotic sex chromosome inactivation.

Figure 1.

ATF7IP2 is highly expressed in male meiosis and accumulates on heterochromatin. (A,C) Heat maps showing bulk RNA-seq gene expression levels across a male germline time course for Atf7ip2 and related genes. (PGC) Primordial germ cells, (ProSG) prospermatogonia, (SG) spermatogonia, (PS) pachytene spermatocytes, (RS) round spermatids. Original data for A are from Seisenberger et al. (2012), Hasegawa et al. (2015), and Maezawa et al. (2018b), and original data for C are from Ishiguro et al. (2020). (B) Track views for MEIOSIN (preleptotene-enriched testes), STRA8 (preleptotene-enriched testes), and RNA polymerase II (POLII; postnatal day 10.5 [P10.5] testes) ChIP-seq data and for CAGE (P10.5 testes). Numbers in brackets indicate ranges of normalized coverage. (D) Schematic of chromosome behavior in meiotic prophase I of male Mus musculus. (Darker green) Autosomes, (lighter green) sex chromosomes. (E) Meiotic chromosome spreads stained with DAPI and antibodies raised against ATF7IP2, SYCP3, and H1T; spreads represent stages of meiotic prophase I. (Insets) H1T immunostaining; H1T is a nuclear marker that appears in mid-pachytene nuclei and persists into haploid spermatids. SYCP3 is a marker of meiotic chromosome axes. Dashed squares are magnified in F. Scale bars, 5 μm. (F, left) Schematic of sex chromosome configuration in male meiosis. (Right) Magnified images of sex chromosomes from E. Scale bars, 5 μm.

Figure 2.

ATF7IP2 is required for male fertility. (A) Schematic of the mouse Atf7ip2 gene and the location of the CRISPR-mediated deletion. (B) Schematic of mouse ATF7IP2 and ATF7IP proteins and their functional domains. (C) Atf7ip2^+/+ and Atf7ip2^−/− males and their testes at postnatal day 66 (P66). Scale bars, 10 mm. (D) Cumulative numbers of pups sired by Atf7ip2^+/− and Atf7ip2^−/− males. (E) Testis weights for Atf7ip2^−/− males and littermate controls (Atf7ip2-ctrl: Atf7ip2^+/+ and Atf7ip2^+/−). Numbers of independent mice analyzed are shown in parentheses. (***) P < 0.001, from pairwise t-tests adjusted with Benjamini–Hochberg post-hoc tests. Data are presented as mean ± SD. (F) Testis sections from Atf7ip2^+/+ and Atf7ip2^−/− mice at 4 mo of age stained with DAPI and antibodies raised against ATF7IP2, γH2AX (a marker of the DNA damage response), and H1T (a marker of germ cells in mid-pachytene and subsequent stages). Scale bars, 100 μm.

Figure 3.

DDR and chromosome synapsis are mildly impaired in Atf7ip2^−/− spermatocytes. (A) Atf7ip2^+/+ and Atf7ip2^−/− spermatocyte chromosome spreads stained with antibodies raised against SYCP3 and γH2AX. γH2AX accumulation patterns are one of three classifications described in C. Scale bars, 10 μm. (B) Meiotic prophase I stage populations quantified as mean ± SD. Numbers of analyzed nuclei are indicated. Data are from three independent littermate pairs at P44, P66, and P69. (*) P < 0.05, from unpaired two-tailed t-tests. (C) Stage-wise proportions of γH2AX accumulation patterns for three independent littermate pairs and are quantified as mean ± SD. Patterns are classified with three criteria (see top). (****) P < 0.0001, from Fisher's exact tests. (D) Chromosome spreads stained with antibodies raised against SYCP3 and MLH1. Arrowheads indicate MLH1 foci. The top dot plot shows distributions of MLH1 counts. The bottom dot plot shows proportions of MLH1 focus-associated XY pseudoautosomal regions (PARS). Numbers of analyzed nuclei are indicated. Data are from three independent littermate pairs at P108, P115, and P122. Bars represent means. P-values are from unpaired t-tests. (E,F) Chromosome spreads stained with antibodies raised against SYCP3 (a marker of all chromosome axes) and SYCP1 (a marker of only synapsed axes). Scale bars: E, 10 μm; F, 5 μm. Bar plots show proportions of pachytene nuclei with normal synapsis of autosomes (E) and sex chromosomes (F). Data are from three independent littermate pairs at P44, P66, and P69 and are presented as mean ± SD. Numbers of analyzed nuclei are indicated. (**) P < 0.01, from unpaired t-tests.

Figure 4.

ATF7IP2 is required for H3K9 methylation on the sex chromosomes during male meiosis. (A) Atf7ip2^+/+ and Atf7ip2^−/− spermatocyte chromosome spreads stained with antibodies raised against H3K9me3 and SYCP3 (a marker of chromosome axes, both synapsed and unsynapsed). Dashed circles indicate the sex chromosomes. Scale bars, 10 μm. (B) H3K9me3 accumulation patterns on the sex chromosomes of Atf7ip2^+/+ and Atf7ip2^−/− early pachytene spermatocytes. Patterns are classified with four criteria (shown at the right). Data are from five independent littermate pairs and are quantified as mean ± SD. Numbers of analyzed nuclei are indicated. (****) P < 0.0001, from a Fisher's exact test. (C) Quantification of mid-pachytene, late pachytene, and diplotene spermatocytes with H3K9me3 signals on the sex chromosomes. Data are from four, six, and four independent littermate pairs, respectively, and are quantified as mean ± SD. Numbers of analyzed nuclei are indicated. (**) P < 0.01, (***) P < 0.001, from unpaired t-tests. (D) Atf7ip2^+/+ and Atf7ip2^−/− chromosome spreads stained with antibodies raised against H3K9me2 and SYCP3. Scale bars, 10 μm. (E) Quantification of diplotene spermatocytes with H3K9me2 signals on the sex chromosomes for four independent experiments quantified as mean ± SD. Numbers of analyzed nuclei are indicated. (***) P < 0.001, from unpaired t-tests. (F) Atf7ip2^+/+ and Atf7ip2^−/− chromosome spreads stained with antibodies raised against SETDB1 and SYCP3. Dashed squares are magnified in the panels at the right. Scale bars, 5 μm. (G) Mdc1^+/+ and Mdc1^−/− spermatocyte chromosome spreads stained with antibodies raised against ATF7IP2 and SYCP3. Scale bars, 10 μm. (H) Summary of the γH2AX/MDC1–ATF7IP2–SETDB1 pathway on X-PCH. (I) Schematic showing the establishment of H3K9me3 on the sex chromosomes in normal mid-pachytene to diplotene spermatocytes.

Figure 5.

scRNA-seq analyses of Atf7ip2^+/+ and Atf7ip2^−/− spermatogenic germ cells. (A) UMAP representations of scRNA-seq transcriptome profiles for germ cells from Atf7ip2^+/+ testes (left, P15), Atf7ip2^−/− testes (middle, P15), and both Atf7ip2^+/+ and Atf7ip2^−/− testes (right). (Gray arrow) Inferred developmental trajectory. (B) Clustering of UMAP-projected scRNA-seq transcriptome profiles for Atf7ip2^+/+ and Atf7ip2^−/− germ cells based on gene expression patterns. (C) Bar graph showing the proportions of Atf7ip2^+/+ and Atf7ip2^−/− germ cells among the clusters. (D) UMAP representations showing expression patterns for key developmental marker genes in spermatogenic cells. Genes shown include Gfra1, which represents undifferentiated spermatogonia; Stra8, which represents differentiating spermatogonia; Meiosin, which represents preleptotene spermatocytes; and Prdm9, which represents early meiotic prophase spermatocytes. (n.s.) Not significant, (*) P < 0.05, from Wilcoxon rank sum tests. (E) Expression patterns for Atf7ip2 and Atf7ip upon the UMAP. (F) Expression levels for autosomal genes. (G) Expression levels for X-chromosomal genes (top) and Y-chromosomal genes (bottom) are shown. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, from Wilcoxon rank sum tests. (H) Summary of Atf7ip2^−/− phenotypes in spermatogenic germ cells. Subtype clusters are ordered by inferred developmental progression. Key cell types and events in Atf7ip2^+/+ and Atf7ip2^−/− spermatogenesis are shown.

Figure 6.

ATF7IP2-binding sites in pachytene spermatocytes. (A) Numbers and genomic distribution of ATF7IP2 CUT&Tag peaks in wild-type pachytene spermatocytes. (B) Clustering analysis of ATF7IP2 CUT&Tag peaks and H3K9me3- and H3K4me3-enriched regions. Average tag density profiles (top) and heat maps (bottom) for each cluster are shown. (C) Chromosomal distribution of ATF7IP2 peak clusters. (D) Genomic distribution of ATF7IP2 peak clusters. (E) Expression levels of ATF7IP2-bound autosomal genes in scRNA-seq. (*) P < 0.05, (**) P < 0.01, from Wilcoxon rank sum tests. (F) Expression levels for ATF7IP2-bound sex chromosomal genes in scRNA-seq. (*) P < 0.05, (***) P < 0.001, from Wilcoxon rank sum tests.

Figure 7.

ATF7IP2 directs meiotic gene regulation and regulates TEs. (A) Comparison of Atf7ip2^+/+ and Atf7ip2^−/− pachytene spermatocyte transcriptomes. Autosomal, X, and Y genes were analyzed separately. Three independent biological replicates were examined. All genes with adjusted P-values (Benjamini–Hochberg method) are plotted. Differentially expressed genes (DEGs; log₂ fold change > 2, adjusted P-value < 0.05) are indicated by color ([red] up-regulated in Atf7ip2^−/−, [blue] down-regulated in Atf7ip2^−/−), and numbers are shown. (B) ATF7IP2 CUT&Tag enrichment at DEG TSSs ± 2 kb in wild-type pachytene spermatocytes. Average tag density profiles (top) and heat maps (bottom) for each cluster are shown. (C) ATF7IP2 CUT&Tag and H3K9me3 CUT&RUN enrichment in clusters I–III (defined in Fig. 6B). Average tag density profiles (top) and heat maps (bottom) for each cluster are shown. (D,E) Track views of the Zfy1 locus (an up-regulated Y-linked locus) and the Hspa2 locus (a down-regulated autosomal locus). (F) Comparison of Atf7ip2^+/+ and Atf7ip2^−/− pachytene spermatocyte transposable element (TE) expression. All TE types are plotted. Differentially expressed TE types (DEGs; log₂ fold change > 2, adjusted P-value < 0.05) are indicated by color ([red] up-regulated in Atf7ip2^−/−, [blue] down-regulated in Atf7ip2^−/−), and numbers are shown. (G) Track view of the ATF7IP2 targeted TEs RLTR10B2 and MMERVK10C-int. (H) Summary and model of the function of ATF7IP2 on X-PCH. (I) Summary and model of the function of ATF7IP2 in TE regulation. (J) Summary and model of the function of ATF7IP2 in gene expression regulation.