ARID1A transcripts detected in pachytene spermatocytes.

scRNA-seq profiles of Arid1a and Arid1b in adult testes from B6 mice (Ernst et al., 2019). (A) tSNE plot describing the clustering of various spermatogenic stages. LOG2 normalized expression counts of (B,C) Arid1a and (D,E) Arid1b represented by tSNE plots and bar plots. (B,D) tSNE plots illustrating the first (tSNE1, x-axis) and second dimensions (tSNE2, y-axis) . (C,E) Bar plots describe the gene’s mRNA abundance (y-axis) across identified germ cell clusters (x-axis). (A) eP: early pachynema, mP: mid pachynema, lP: late pachynema, MI: Metaphase-I, MII: Metaphase-II, S1-S11: Spermatid stages. Plots generated with shiny app (https://marionilab.cruk.cam.ac.uk/SpermatoShiny)

ARID1A associates with the sex body late in meiotic prophase-I.

Representative pachytene and diplotene spermatocyte spreads immunolabelled for ARID1A (magenta) and SYCP3 (green) and then counterstained for DNA (blue). Scale bar:10 μm, magnification: 100x.

Spermatogenesis appears unperturbed in Arid1acKO males.

Histological examination of (A) PAS-stained seminiferous tubule sections and (B) H&E-stained cauda epididymal sections obtained from 10-week-old Arid1aWT and Arid1acKO mice. The micrograph depicts seminiferous tubule staging and scale bars (50 μm). Magnification: 40x.

Arid1acKOtestes display inefficient Stra8-Cre activity.

(A) Schematic (top) illustrating the region of Arid1aflallele spanning exons (ex) 4-8 (grey boxes) along with the location of loxP sites (solid magenta arrowheads) and primer annealing sites (blue arrows). (A bottom): analysis of the RT-PCR of Arid1a transcripts from STA-PUT purified populations of Arid1aWT and Arid1acKO pachytene spermatocytes (left) and round spermatids (right). DNA electrophoresis gels indicate bands corresponding to the fl: floxed and Δ: excised PCR products. Arid1aWTand Arid1acKO (B) testes cryosections, and (C) spermatocyte spreads, immunolabelled for ARID1A (magenta), SYCP3 (green), and counterstained for DNA (blue). (B) Scale bar: 20 μm, magnification: 63x, P: Pachytene, D: Diplotene, and M-I: Metaphase-I spermatocytes. (C) Scale bar:15 μm, magnification: 100x.

The loss of ARID1A results in a pachytene arrest.

Table outlining the distribution of P23 Arid1aWT and Arid1acKOmeiotic prophase-I profiles. SYCP3 staining determined meiotic staging. Co-staining of ARID1A identified mutant spermatocytes from internal controls. The total number of spermatocytes scored included 166 for Aridafl/fland 124 for Arid1acKO. * denotes Arid1acKO diplotene spermatocytes with partially reduced but not complete loss of ARID1A signal relative to controls.

Requirement of ARID1A for the repression of sex-linked genes.

(A) MA plot describing the LOG2-fold-change (LFC, y-axis) in mean expression (x-axis) of genes displaying significant (FDR≤0.05, magenta dots) changes in Arid1acKO relative to Arid1aWT pachytene spermatocytes. Dashed blue lines denote 2 LFC. Gray dots (FDR≥0.05) depict non-significant changes in gene expression. (B) Violin plot describing the LOG2-fold-change (LFC, y-axis) in the median chromosome-wide gene expression (x-axis) in Arid1acKO relative to Arid1aWT pachytene spermatocytes. The dashed blue line denotes no change in gene expression.

Transcription start sites of differentially expressed autosomal and sex-linked genes display ARID1A occupancy.

(A-B) Heatmap (bottom) and metaplot (top) displaying the average gene-wide enrichment of ARID1A associated with differentially expressed (A) autosomal and (B) sex-linked genes in Arid1aWTand Arid1acKO pachytene spermatocytes. (A-B) The number of RefSeq annotations (n) associated with differentially regulated autosomal and sex-linked genes (rows) is indicated. Up-reg: misexpressed and Dn-reg: misrepressed genes in response to the loss of ARID1A. Average ARID1A CUT&RUN coverage was determined using two antibodies (anti-ARID1ASigma; anti-ARID1ACST) plotted across RefSeq genes ± 3Kb.

ARID1A does not influence sex body formation.

(A) Arid1aWTand Arid1acKO spermatocyte spreads immunolabelled for ARID1A (magenta) and γH2Ax (green). Scale bar:15 μm, magnification: 100x. (B) Cross section of adult (3-month-old) Arid1aWTand Arid1acKO seminiferous tubules immunolabelled for ARID1A (green) and ATR (cyan). Scale bar:15 μm, magnification: 63x. (C) Arid1aWTand Arid1acKO pachytene spermatocytes immunolabelled for MDC1 (magenta), SYCP3 (cyan), and ARID1A (green). Scale bar:15 μm, magnification: 100x. (A-B) DNA counterstained with DAPI (blue).

ARID1A limits RNA polymerase II (RNAPII) localization to the sex body.

(A) Arid1aWTand Arid1acKO pachytene spermatocytes immunolabelled for pSer2-RNAPII (green), SYCP3 (cyan), and counterstained with DAPI (blue). Scale bar:15 μm, magnification: 100x. The sex chromosomes (white arrow) and sex body (yellow dashed circle) are labeled. (B) Dot plot describing the corrected total pSer2-RNAPII fluorescence (y-axis) measured from Arid1aWT (n = 82) and Arid1acKO (n= 119) pachytene spermatocytes (3 replicates per genotype). Empty diamonds (magenta) represent independent data points. Significance determined by a two-tailed unpaired Student’s t-test p values. Data expressed as mean (black dot) ± SEM.

ARID1A limits promoter accessibility.

(A) Genomic associations of MACS2 derived ATAC-seq peak calls from Arid1aWT and Arid1acKO pachytene spermatocytes. Percent (%) distribution of genomic annotations is indicated. (B-C) Heatmap (bottom) and metaplot (top) displaying the average ATAC-seq signal associated with differentially expressed (B) autosomal and (C) sex-linked genes in Arid1aWTand Arid1acKO pachytene spermatocytes. (B-C) The number of RefSeq annotations (n) associated with differentially regulated genes (rows) is indicated. Up-reg: misexpressed, and Dn-reg: misrepressed genes in response to the loss of ARID1A. Average ATAC-seq coverage was plotted across RefSeq genes ± 3Kb.

ARID1A influences the chromatin composition of the sex body.

(A-B) Arid1aWT and Arid1acKO pachytene spermatocytes immunolabelled for HORMAD1 (magenta), (A) ARID1A (cyan) and H3.3 (green), (B) ARID1A (green) and H3.1/3.2 (cyan). (A-B) Proportion (%) of Arid1aWT and Arid1acKO early, mid, and late pachytene spermatocytes displaying distinct (A) H3.3, (B) H3.1/3.2 localization patterns with the sex body. For H3.3 immunostaining, the total number of Arid1aWT pachytene spermatocytes: early = 144, mid= 274, late= 95; Arid1acKO pachytene spermatocytes: early = 43, mid= 86, late= 55, were scored, from 3 replicates each. For H3.1/3.2 immunostaining, the total number of Arid1aWT pachytene spermatocytes: early = 91, mid= 124, late= 45; Arid1acKO pachytene spermatocytes: early = 22, mid= 116, late= 58, were scored, from 3 replicates each. DNA counterstained with DAPI (blue). The sex chromosomes (yellow arrow) and sex body (yellow dashed circle) are labeled. Scale bar:15 μm, magnification: 100x.

ARID1A does not influence the expression or incorporation of H3.3.

(A) Western blots on acid-extracted histones obtained from four independent replicates of P19 Arid1aWT and Arid1acKO spermatogenic cells, displaying H3.3 abundance. Total histone levels from each sample are displayed. (B) Arid1aWT and Arid1acKO pachytene spermatocytes immunolabelled for HORMAD1 (magenta), DAXX (cyan), and ARID1A (green). DNA counterstained with DAPI (blue). The sex chromosomes (yellow arrow) and sex body (yellow dashed circle) are labeled. Scale bar:15 μm, magnification: 100x. (C) Arid1aWTand Arid1acKO testis cryosections immunolabelled for ARID1A (magenta) and HIRA (green). DNA counterstained with DAPI (blue). Representative mutant (white arrows) and internal control (yellow arrows) pachytene spermatocytes labeled in Arid1acKO testis cryosection. Scale bar: 15 μm, magnification: 63x.

ARID1A limits H3.3 occupancy at promoters

(A) Genomic associations of MACS2 H3.3 peak calls from Arid1aWT and Arid1acKO pachytene spermatocytes. Percent (%) distribution of genomic annotations is indicated. (B-C) Heatmap (bottom) and metaplot (top) displaying the average H3.3 signal associated with differentially expressed (B) sex-linked and (C) autosomal genes in Arid1aWT and Arid1acKO pachytene spermatocytes. (B-C) The number of RefSeq annotations (n) associated with differentially regulated genes (rows) are indicated. Up-reg: misexpressed, and Dn-reg: misrepressed genes in response to the loss of ARID1A. Average H3.3 coverage plotted across RefSeq genes ± 3Kb.

Genome browser views of ARID1A governed H3.3 genomic associations.

H3.3 CUT&RUN coverage from Arid1aWT (green tracks) and Arid1acKO(Orange tracks) pachytene spermatocytes. Solid red bars and yellow highlights denote H3.3 peak calls (MACS2) from Arid1aWT and Arid1acKOpachytene spermatocytes. Vertical viewing limits within parentheses.

H3.3 displays differential occupancy across ARID1A governed sex-linked open chromatin.

(A-B) Heatmaps (bottom) and metaplots (top) displaying average (A) chromatin accessibility (purple heatmap) and H3.3 enrichment (green heatmap) associated with lost, common, and gained ATAC-seq peak calls (MACS2), (B) enrichment of H3.3 at k-means clusters associated with common (C1-C4, left) and gained (G1-G4, right) ATAC-seq peaks, in Arid1aWTand Arid1acKO pachytene spermatocytes. (A-B) ATAC-seq and H3.3 coverage plotted over a 5 Kb window centered at ATAC-seq peaks (MACS2). Number of ATAC-seq peak calls (n) associated with each category is indicated.

H3.3 occupancy is antagonistic to DMC1 associations in non-homologous sex-linked regions.

(A) Genomic associations of common (C1-C4) and gained (G1-G4) k-means clusters in pachytene spermatocytes. Percent (%) distribution of genomic annotations is indicated. (B) Results of PRDM9 motif enrichment analyses at gained (G1 and G3), k-means clusters (top), and plots describing the frequency of PRDM9 motifs (y-axis) spanning a 5 Kb window centered at ATAC-seq peaks (x-axis) associated with gained (middle) and common (bottom) k-means clusters that are either deficient (orange trend line) or enriched (cyan trend line) for H3.3 occupancy. Trend lines were generated using generalized additive mode smoothing (gam). 95% confidence intervals (gray shading) are indicated. (C-D) Heatmaps displaying testes DMC1 (left) and pachytene spermatocyte associated H3K4me3 (right) enrichment relative to input at k-means clusters associated with (C) gained (G1-G4), and (D) common (C1-C4) ATAC-seq peaks. (C-D) DMC1 and H3K4me3 coverage plotted over a 10 Kb window centered at ATAC-seq peaks (MACS2).

ARID1A influences the axial association of DMC1 with the XY during pachynema.

(A) Arid1aWTand Arid1acKO pachytene spermatocytes immunolabelled for HORMAD1 (magenta), ARID1A (green), DMC1 (cyan), and counterstained with DAPI (blue). Scale bar:15 μm, magnification: 100x. A magnified view of the sex chromosomes is indicated (yellow dashed square). (B) Dot plot displaying the number of sex-linked DMC1 foci (y-axis) quantified from Arid1aWT (n = 66) and Arid1acKO(ARID1A-, n= 75) pachytene spermatocytes, obtained from 3 replicates each. Empty diamonds (magenta) represent independent data points. Significance determined using a two-tailed unpaired Student’s t-test p values. Data expressed as mean (black dot) ± SEM.

ARID1A does not affect the axial association of RAD51 with the XY during pachynema.

(A) Heatmaps displaying testes RAD51 enrichment relative to input at k-means clusters associated with gained (G1-G4, left) and common (C1-C4, right) ATAC-seq peaks. RAD51 coverage plotted over a 10 Kb window centered at ATAC-seq peaks (MACS2). (B) Arid1aWT and Arid1acKO pachytene spermatocytes immunolabelled for HORMAD1 (magenta), RAD51 (green) and counterstained with DAPI (blue). Scale bar:15 μm, magnification: 100x. Yellow arrows and dashed circles label the sex chromosomes.

A model describing the role of ARID1A sex-linked chromatin regulation.

During meiosis, the sex chromosomes undergo transcriptional repression at the onset of pachynema. A dramatic change in the composition of sex-linked chromatin accompanies this chromosome-wide repression. From mid to late pachynema, spermatocytes display a hyper-accumulation of the variant histone H3.3 (Ochre shading and gradient) on the sex chromosomes (magenta) relative to autosomes (green). Concomitantly, the levels of the canonical histones H3.1/3.2 and elongating pSer2-RNAPII complex (grey gradients) appear depleted from the sex body by late pachynema. The loss of ARID1A dramatically alters the chromatin composition of the sex body, which features low H3.3 (yellow bar) association at levels indistinguishable from autosomes throughout pachynema. Concomitantly, canonical H3.1/3.2 and pSer2-RNAPII levels (grey bar) on the sex body remain abnormally stable throughout pachynema. These sex-linked chromatin aberrations, along with persistent transcription owing to the association of pSer2-RNAPII with mutant sex body, fail meiotic sex chromosome inactivation (MSCI) and, consequently, pachytene arrest. This defect also coincides with an abnormal loss of DMC1 (blue foci) localization to the unpaired sex chromatids in response to the loss of ARID1A. Therefore, along with transcriptional repression, ARID1A-governed chromatin dynamics appear to influence DNA repair on the sex chromosomes.