ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse

Debashish U. Menon
Prabuddha Chakraborty
Noel Murcia
Terry Magnuson author has email address

Department of Genetics, and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7264, USA

https://doi.org/10.7554/eLife.88024.3

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Figures and data

ARID1A associates with the sex body late in meiotic prophase-I.
Representative wild-type pachytene and diplotene spermatocyte spreads immunolabelled for ARID1A (magenta) and SYCP3 (green) and then counterstained for DNA (blue). Panel insets denote a magnified view of the nuclear area surrounding the sex chromosomes. Scale bar:10 μm, magnification: 100x

The loss of ARID1A results in a pachytene arrest.
Table outlining the distribution of P23 Arid1a^WT and Arid1a^cKO meiotic prophase-I profiles. SYCP3 staining determined meiotic staging. Co-staining of ARID1A identified mutant spermatocytes from escaper. The total number of spermatocytes scored included 166 for Arida^fl/fl and 124 for Arid1a^cKO. * denotes Arid1a^cKO 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 Arid1a^cKO relative to Arid1a^WT 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 Arid1a^cKO relative to Arid1a^WT pachytene spermatocytes. The dashed blue line denotes no change in gene expression.

ARID1A limits RNA polymerase II (RNAPII) localization to the sex body.
(A) Arid1a^WT and Arid1a^cKO 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. Brightness of the whole image panel (A) was increased by compressing the dynamic range of the image panel using Adobe photoshop (0-1-255 to 0-1-150). (B) Dot plot describing the corrected total pSer2-RNAPII fluorescence (y-axis) measured from Arid1a^WT (n = 82) and Arid1a^cKO (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 Arid1a^WT and Arid1a^cKO 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 Arid1a^WTand Arid1a^cKO 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) Arid1a^WT and Arid1a^cKO 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 Arid1a^WTand Arid1a^cKO 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 Arid1a^WT pachytene spermatocytes: early = 144, mid= 274, late= 95; Arid1a^cKO pachytene spermatocytes: early = 43, mid= 86, late= 55, were scored, from 3 replicates each. For H3.1/3.2 immunostaining, the total number of Arid1a^WTpachytene spermatocytes: early = 91, mid= 124, late= 45; Arid1a^cKOpachytene 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. Brightness of the whole image panel (Fig. 5) was increased by compressing the dynamic range of the image panels using Adobe photoshop (0-1-255 to 0-1-150).

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 Arid1a^WT and Arid1a^cKOpachytene 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.

ARID1A influences the axial association of DMC1 with the XY during pachynema.
(A) Arid1a^WT and Arid1a^cKO 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 Arid1a^WT (n = 66) and Arid1a^cKO (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.

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 canonical histones H3.1/3.2 levels 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.

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