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

Reviewer #1 (Public Review):

The work by Debashish U. Menon, Noel Murcia, and Terry Magnuson brings important knowledge about histone H3.3 dynamics involved in meiotic sex chromosome inactivation (MSCI). MSCI is unique to gametes and failure during this process can lead to infertility. Classically, MSCI has been studied in the context of DNA Damage repair pathways and little is known about the epigenetic mechanisms behind maintenance of the sex body as a silencing platform during meiosis. One of the major strengths of this work is the evidence provided on the role of ARID1A, a BAF subunit, in MSCI through the regulation of H3.3 occupancy in specific genic regions.

Using RNA seq and CUT&RUN and ATAC-seq, the authors show that ARID1A regulates chromatin accessibility of the sex chromosomes and XY gene expression. Loss of ARID1A increases promoter accessibility of XY linked genes with concomitant influx of RNA pol II to the sex body and up regulation of XY-linked genes. This work suggests that ARID1A regulates chromatin composition of the sex body since in the absence of ARID1A, spermatocytes show less enrichment of H3.3 in the sex chromosomes and stable levels of the canonical histones H3.1/3.2. By overlapping CUT&RUN and ATAC-seq data, authors show that changes in chromatin accessibility in the absence of ARID1A are given by redistribution of occupancy of H3.3. Gained open chromatin in mutants corresponds to up regulation of H3.3 occupancy at transcription start sites of genes mediated by ARID1A.

Interestingly, ARID1A loss caused increased promoter occupancy by H3.3 in regions usually occupied by PRDM9. PRDM9 catalyzes histone H3 lysine 4 trimethylation during meiotic prophase I, and positions double strand break (DSB) hotspots. Lack of ARID1A causes reduction in occupancy of DMC1, a recombinase involved in DSB repair, in non-homologous sex regions. These data suggest that ARID1A might indirectly influence DNA DSB repair on the sex chromosomes by regulating the localization of H3.3. This is very interesting given the recently suggested role for ARID1A in genome instability in cancer cells. It raises the question of whether this role is also involved in meiotic DSB repair in autosomes and/or how this mechanism differs in sex chromosomes compared to autosomes.

The fact that there are Arid1a transcripts that escape the Cre system in the Arid1a KO mouse model might difficult the interpretation of the data. The phenotype of the Arid1a knockout is probably masked by the fact that many of the sequencing techniques used here are done on a heterogeneous population of knockout and wild type spermatocytes. In relation to this, I think that the use of the term "pachytene arrest" might be overstated, since this is not the phenotype truly observed. Nonetheless, the authors provide evidence showing that the spermatids observed in cKO testes that progress in spermatogenesis are the ones expressing Arid1a. This work presents enough evidence to include the BAF complex as part of the MSCI process, which increases our knowledge on specific regulation of the sex chromatin during meiosis.

Reviewer #2 (Public Review):

The authors tried to characterize the function of the SWI/SNF remodeler family, BAF, in spermatogenesis. The authors focused on ARID1A, a BAF-specific putative DNA binding subunit, based on gene expression profiles.

The authors disagreed with my previous assessments. I disagree with their response.

Reviewer #3 (Public Review):

In this manuscript, Magnuson and colleagues investigate the meiotic functions of ARID1A, a putative DNA binding subunit of the SWI/SNF chromatin remodeler BAF. The authors develop a germ cell specific conditional knockout (cKO) mouse model using Stra8-cre and observe that ARID1A-deficient cells fail to progress beyond pachytene, although due to inefficiency of the Stra8-cre system the mice retain ARID1A-expressing cells that yield sperm and allow fertility. Because ARID1A was found to accumulate at the XY body late in Prophase I, the authors suspected a potential role in meiotic silencing and by RNAseq observe significant misexpression of sex-linked genes that typically are silenced at pachytene. They go on to show that ARID1A is required for exclusion of RNA PolII from the sex body and for limiting promoter accessibility at sex-linked genes, consistent with a meiotic sex chromosome inactivation (MSCI) defect in cKO mice. The authors proceed to investigate the impacts of ARID1A on H3.3 deposition genome-wide. H3.3 is known be regulated by ARID1A and is linked to silencing, and here the authors find that upon loss of ARID1A, overall H3.3 enrichment at the sex body as measured by IF failed to occur, but H3.3 was enriched specifically at transcriptional start sites of sex-linked genes that are normally regulated by ARID1A. The results suggest that ARID1A normally prevents H3.3 accumulation at target promoters on sex chromosomes and based on additional data, restricts H3.3 to intergenic sites. Finally, the authors present data implicating ARID1A and H3.3 occupancy in DSB repair, finding that ARID1A cKO leads to a reduction in focus formation by DMC1, a key repair protein. Overall the paper provides new insights into the process of MSCI from the perspective of chromatin composition and structure, and raises interesting new questions about the interplay between chromatin structure, meiotic silencing and DNA repair.

In general the data are convincing. The conditional KO mouse model has some inherent limitations due to incomplete recombination and the existence of 'escaper' cells that express ARID1A and progress through meiosis normally. This reviewer feels that the authors have addressed this point thoroughly and have demonstrated clear and specific phenotypes using the best available animal model. The data demonstrate that the mutant cells fail to progress past pachytene, although it is unclear whether this specifically reflects pachytene arrest, as accumulation in other stages of Prophase also is suggested by the data in Table 1.

The revised manuscript more appropriately describes the relationship between ARID1A and DNA damage response (DDR) signaling. The authors don't see defects in a few DDR markers in ARID1A CKO cells (including a low resolution assessment of ATR), suggesting that ARID1A may not be required for meiotic DDR signaling. However, as previously noted the data do not rule out the possibility that ARID1A is downstream of DDR signaling, and the authors note the possibility of a role for DDR signaling upstream of ARID1A.

A final comment relates to the impacts of ARID1A loss on DMC1 focus formation and the interesting observation of reduced sex chromosome association by DMC1. The authors additionally assess the related recombinase RAD51 and suggest that it is unaffected by ARID1A loss. However, only a single image of RAD51 staining in the cKO is provided (Fig. S11) and there are no associated quantitative data provided. The data are suggestive and conclusions about the impacts of ARID1A loss on RAD51 must be considered as preliminary until more rigorously assessed.

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews

Reviewer #1 (Public Review):

Comment: The fact that there are Arid1a transcripts that escape the Cre system in the Arid1a KO mouse model might difficult the interpretation of the data. The phenotype of the Arid1a knockout is probably masked by the fact that many of the sequencing techniques used here are done on a heterogeneous population of knockout and wild type spermatocytes. In relation to this, I think that the use of the term "pachytene arrest" might be overstated, since this is not the phenotype truly observed. Knockout mice produce sperm, and probably litters, although a full description of the subfertility phenotype is lacking, along with identification of the stage at which cell death is happening by detection of apoptosis.

Response: As the reviewer indicates, we did not observe a complete arrest at Pachynema. In fact, the histology shows the presence of spermatids and sperm in seminiferous tubules and epididymides (Fig. Sup. 3). However, our data argue that the wild-type haploid gametes produced were derived from spermatocyte precursors that have likely escaped Cre mediated activity (Fig. Sup. 4). Furthermore, diplotene and metaphase-I spermatocytes lacking ARID1A protein by IF were undetectable in the Arid1acKO testes (Fig. S4B). Therefore, although we do not demonstrate a strict pachytene arrest, it is reasonable to conclude that ARID1A is necessary to progress beyond pachynema. We have revised the manuscript to reflect this point (Abstract lines 17,18; Results lines 153,154)

Comment: It is clear from this work that ARID1a is part of the protein network that contributes to silencing of the sex chromosomes. However, it is challenging to understand the timing of the role of ARID1a in the context of the well-known DDR pathways that have been described for MSCI.

Response: With respect to the comment on the lack of clarity as to which stage of meiosis we observe cell death, our data do suggest that it is reasonable to conclude that mutant spermatocytes (ARID1A-) undergo cell death at pachynema given their inability to execute MSCI, which is a well-established phenotype.

Comment: Staining of chromosome spreads with Arid1a antibody showed localization at the sex chromosomes by diplonema; however, analysis of gene expression in Arid1a KO was performed on pachytene spermatocytes. Therefore, is not very clear how the chromatin remodeling activity of Arid1a in diplonema is affecting gene expression of a previous stage. CUTnRUN showed that ARID1a is present at the sex chromatin in earlier stages, leading to hypothesize that immunofluorescence with ARID1a antibody might not reflect ARID1a real localization.

Response: It is unclear what the reviewer means about not understanding how ARID1A activity at diplonema affects gene expression at earlier stages. Our interpretations were not based solely on the observation of ARID1A associations with the XY body at diplonema. In fact, mRNA expression and CUT&RUN analyses were performed on pachytene-enriched populations. ARID1A's association with the XY body is not exclusive to diplonema. Based on both CUT&RUN and IF data, ARID1A associates with XY chromatin as early as pachynema. Only at late diplonema did we observe ARID1A hyperaccumulation on the XY body by IF.

Reviewer #2 (Public Review):

Comment: The inefficient deletion of ARID1A in this mouse model does not allow any detailed analysis in a quantitative manner.

Response: As explained in our response to these comments in the first revision, we respectfully disagree with this reviewer’s conclusions. We have been quantitative by co-staining for ARID1A, ensuring that we can score mutant pachytene spermatocytes from escapers. Additionally, we provide data to show the efficiency of ARID1A loss in the purified pachytene populations sampled in our genomic assays.

Reviewer #3 (Public Review):

Comment: The data demonstrate that the mutant cells fail to progress past pachytene, although it is unclear whether this specifically reflects pachytene arrest, as accumulation in other stages of Prophase also is suggested by the data in Table 1. The western blot showing ARID1A expression in WT vs. cKO spermatocytes (Fig. S2) is supportive of the cKO model but raises some questions. The blot shows many bands that are at lower intensity in the cKO, at MWs from 100-250kDa. The text and accompanying figure legend have limited information. Are the various bands with reduced expression different isoforms of ARID1A, or something else? What is the loading control 'NCL'? How was quantification done given the variation in signal across a large range of MWs?

Response: The loading control is Nucleolin. With respect to the other bands in the range of 100-250 kDa, it is difficult to say whether they represent ARID1A isoforms. The Uniprot entry for Mouse ARID1A only indicates a large mol. wt sequence of ~242 kDa; therefore, the band corresponding to that size was quantified. There is no evidence to suggest that lower molecular weight isoforms may be translated. Although speculative, it is possible that the lower molecular weight bands represent proteolytic/proteasomal degradation products or products of antibody non-specificity. These points are addressed in the revised manuscript (Legend to Fig S2, lines 926-931). Blots were scanned on a LI-COR Odyssey CLx imager and viewed and quantified using Image Studio Version 5.2.5 (Methods, lines 640-642).

Comment: An additional weakness relates to how the authors describe the relationship between ARID1A and DNA damage response (DDR) signaling. The authors don't see defects in a few DDR markers in ARID1A CKO cells (including a low-resolution assessment of ATR), suggesting that ARID1A may not be required for meiotic DDR signaling. However, as previously noted the data do not rule out the possibility that ARID1A is downstream of DDR signaling and the authors even indicate that "it is reasonable to hypothesize that DDR signaling might recruit BAF-A to the sex chromosomes (lines 509-510)." It therefore is difficult to understand why the authors continue to state that "...the mechanisms underlying ARID1A-mediated repression of the sex-linked transcription are mutually exclusive to DDR pathways regulating sex body formation" (p. 8) and that "BAF-A-mediated transcriptional repression of the sex chromosomes occurs independently of DDR signaling" (p. 16). The data provided do not justify these conclusions, as a role for DDR signaling upstream of ARID1A would mean that these mechanisms are not mutually exclusive or independent of one another.

Response: The reviewer’s argument is reasonable, and we have made the recommended changes (Results, lines 212-215; Discussion, lines 499-500).

Comment: A final comment relates to the impacts of ARID1A loss on DMC1 focus formation and the interesting observation of reduced sex chromosome association by DMC1. The authors additionally assess the related recombinase RAD51 and suggest that it is unaffected by ARID1A loss. However, only a single image of RAD51 staining in the cKO is provided (Fig. S11) and there are no associated quantitative data provided. The data are suggestive but it would be appropriate to add a qualifier to the conclusion regarding RAD51 in the discussion which states that "...loss of ARID1a decreases DMC1 foci on the XY chromosomes without affecting RAD51" given that the provided RAD51 data are not rigorous. In the long-term it also would be interesting to quantitatively examine DMC1 and RAD51 focus formation on autosomes as well.

Response: We agree with the reviewer’s comment and have made the recommended changes (Discussion, lines 518-519).

Response to non-public recommendations

Reviewer 2:

Comment: Meiotic arrest is usually judged based on testicular phenotypes. If mutant testes do not have any haploid spermatids, we can conclude that meiotic arrest is a phenotype. In this case, mutant testes have haploid spermatids and are fertile. The authors cannot conclude meiotic arrest. The mutant cells appear to undergo cell death in the pachytene stage, but the authors cannot say "meiotic arrest."

Response: We disagree with this comment. By IF, we see that ~70% of the spermatocytes have deleted ARID1A. Furthermore, we never observed diplotene spermatocytes that lacked ARID1A. The conclusion that the absence of ARID1A results in a pachynema arrest and that the escapers produce the haploid spermatids is firm.

Comment: Fig. S2 and S3 have wrong figure legends.

Response: The figure legends for Fig. S2 and S3 are correct.

Comment: The authors do not appear to evaluate independent mice for scoring (the result is about 74% deletion above, Table S1). Sup S2: how many independent mice did the authors examine?

Response:These were Sta-Put purified fractions obtained from 14-15 WT and mutant mice. It is difficult to isolate pachytene spermatocytes by Sta-Put at the required purity in sufficient yields using one mouse at a time. We used three technical replicates to quantify the band intensity, and the error bars represent the standard error of the mean (S.E.M) of the band intensity.

Comment: Comparison of cKO and wild-type littermate yielded nearly identical results (Avg total conc WT = 32.65 M/m; Avg total conc cKO = 32.06 M/ml)". This sounds like a negative result (i.e., no difference between WT and cKO).

Response: This is correct. There is no difference between Arid1aWT and Arid1aCKO sperm production. This is because wild-type haploid gametes produced were derived from spermatocyte precursors that have escaped Cre-mediated activity (Fig. S4). These data merely serve to highlight an inherent caveat of our conditional knockout model and are not intended to support the main conclusion that ARID1A is necessary for pachytene progression.

Comment: The authors now admit ~ 70 % efficiency in deletion, and the authors did not show the purity of these samples. If the purity of pachytene spermatocytes is ~ 80%, the real proportion of mutant cells can be ~ 56%. It is very difficult to interpret the data.

Response: The original submission did refer to inefficient Cre-induced recombination. The reviewer asked for the % efficiency, which was provided in the revised version. Also, please refer to Fig. S2, where Western blot analysis demonstrates a significant loss of ARID1A protein levels in CKO relative to WT pachytene spermatocyte populations that were used for CUT&RUN data generation.

Comment: The authors should not use the other study to justify their own data. The H3.3 ChIP-seq data in the NAR paper detected clear peaks on autosomes. However, in this study, as shown in Fig. S7A, the authors detected only 4 peaks on autosomes based on MACS2 peak calling. This must be a failed experiment. Also, S7A appears to have labeling errors.

Response: I believe the reviewer is referring to supplementary figure 8A. Here, it is not clear which labeling errors the reviewer is referring to. In the wild type, the identified peaks were overwhelmingly sex-linked intergenic sites. This is consistent with the fact that H3.3 is hyper-accumulated on the sex chromosomes at pachynema.

The authors of the NAR paper did not perform a peak-calling analysis using MACS2 or any other peak-calling algorithm. They merely compared the coverage of H3.3 relative to input. Therefore, it is not clear on what basis the reviewer says that the NAR paper identified autosomal peaks. Their H3.3 signal appears widely distributed over a 6 kb window centered at the TSS of autosomal genes, which, compared to input, appears enriched. Our data clearly demonstrates a less noisy and narrower window of H3.3 enrichment at autosomal TSSs in WT pachytene spermatocytes, albeit at levels lower than that seen in CKO pachytene spermatocytes (Fig S8B and see data copied below for each individual replicate). Moreover, the lack of peaks does not mean that there was an absence of H3.3 at these autosomal TSSs (Supp. Fig. S8B). Therefore, we disagree with the reviewer’s comment that the H3.3 CUT&RUN was a failed experiment.

Author response image 1.

H3.3 Occupancy at genes mis-regulated in the absence of ARID1A

Comment: If the author wishes to study the function of ARID2 in spermatogenesis, they may need to try other cre-lines to have more robust phenotypes, and all analyses must be redone using a mouse model with efficient deletion of ARID2.

Response: As noted, we chose Stra8-Cre to conditionally knockout Arid1a because ARID1A is haploinsufficient during embryonic development. The lack of Cre expression in the maternal germline allows for transmission of the floxed allele, allowing for the experiments to progress.

Comment: The inefficient deletion of ARID1A in this mouse model does not allow any detailed analysis in a quantitative manner.

Response: In many experiments, we have been quantitative when possible by co-staining for ARID1A, ensuring that we can score mutant pachytene spermatocytes from escapers. Additionally, we provide data to show the efficiency of ARID1A loss in the purified pachytene populations sampled in our genomic assays.

Reviewer 3:

Comment: The Methods section refers to antibodies as being in Supplementary Table 3, but the table is labeled as Supplementary Table 2.

Response: This has been corrected