Chromosome-specific maturation of the epigenome in the Drosophila male germline

Reviewer #1 (Public Review):

Anderson, Henikoff, Ahmad et al. performed a series of genomics assays to study Drosophila spermatogenesis. Their main approaches include (1) Using two different genetic mutants that arrest male germ cell differentiation at distinct stages, bam and aly mutant, they performed CUT&TAG using H3K4me2, a histone modification for active promoters and enhancers; (2) Using FACS sorted pure spermatocytes, they performed CUT&TAG using antibodies against RNA PolII phosphorylated Ser 2, H4K16ac, H3K9me2, H3K27me3, and ubH2AK118. They also compare these chromatin profiling results with the published single-cell and single-nucleus RNA-seq data. Their analyses are across the genome but the major conclusions are about the chromatin features of the sex chromosomes. For example, the X chromosome is lack of dosage compensation as well as inactivation in spermatocytes, while Y chromosome is activated but enriched with ubH2A in spermatocytes. Overall, this work provides high-quality epigenome data in testes and in purified germ cells. The analyses are very informative to understand and appreciate the dramatic chromatin structure change during spermatogenesis in Drosophila. Some new analyses and a few new experiments are suggested here, which hopefully further take advantage of these data sets and make some results more conclusive.

Major comments:

1). The step-wise accumulation of H3K4me2 in bam, aly and wt testes are interesting. Is it possible to analyse the cis-acting sequences of different groups of genes with distinct H3K4me2 features, in order to examine whether there is any shared motif(s), suggesting common trans-factors that potentially set up the chromatin state for activating gene expression in a sequential manner?

2). Pg. 4, line 141-142: "we cannot measure H3K4me2 modification at the bam promoter in bam mutant testes or at the aly promoter in aly mutant testes", what are the allelic features of the bam mutant and aly mutant? Are the molecular features of these mutations preventing the detection of H3K4me2 at the endogenous genes' promoters? Also, the references cited (Chen et al., 2011) and (Laktionov et al., 2018) are not the original research papers where these two mutants were characterized.

3). The original paper that reported the Pc-GFP line and its localization is: Chromosoma 108, 83 (1999). The Pc-GFP is ubiquitously expressed and almost present in all cell types. In Figure 6B, there is no Pc-GFP signals in bam and aly mutant cells. According to the Method "one testis was dissected", does it mean that only one testis was prepared for immunostaining and imaging? If so, definitely more samples should be used for a more confident conclusion. Also, why use 3rd instar larval testes instead of adult testes? Finally, it is better to compare fixed tissue and live tissue, as the Pc-GFP signal could be lost during fixation and washing steps. Please refer to the above paper [Chromosoma 108, 83 (1999)] for Pc-GFP in spermatogonial cells and Development 138, 2441-2450 (2011) for Pc-GFP localization in aly mutant.

4). Ubiquitinylation of histone H2A is typically associated with gene silencing, here it has been hypothesized that ubH2A contributes to the activation of Y chromosome. This conclusion is strenuous, as it entirely depends on correlative results. For example, the lack of co-localization of ubH2A immunostaining and Pc-GFP are not convincing evidence that ubH2A is not resulting from PRC1 dRing activity. It would be a lot stronger conclusion by using genetic tools to show this. For example, if dRing is knocked down (using RNAi driven by a late-stage germline driver such as bam-Gal4) or mutated in spermatocytes (using mitotic clonal analysis), would they detect changes of ubH2A levels?

5). Regarding "X chromosome of males is thought to be upregulated in early germline cells", it has been shown that male-biased genes are deprived on the X chromosome [Science 299:697-700 (2003); Genome Biol 5:R40 (2004); Nature 450:238-241 (2007)], so are the differentiation genes of spermatogenesis [Cell Research 20:763-783 (2010)]. It would be informative to discuss the X chromatin features identified in this work with these previous findings. For example, the lack of RNAPII on X chromosome in spermatocytes could be due to a few differentiation genes expressed in spermatocytes located on the X chromosome.

Reviewer #2 (Public Review):

Anderson et al profiled chromatin features, including active chromatin marks, RNA polymerase II distribution, and histone modifications in the sex chromosomes of spermatogenic cells in Drosophila. The results are new and the experiments and analyses look well done, including with appropriate numbers of replicates. Results were parsed by comparing them among two arrest mutants and wildtype, as well as in FACS-sorted spermatocytes. The authors also profiled larval wing discs to serve as reference-somatic cells, which allowed them to focus only on features in their testis data that were associated with germ cells. Their results were further refined by categorizing the genes of interest based on available single nucleus RNA seq expression profiles. The authors document interesting phenomena, such as differences in the distribution of RNAPIIS2p on some genes in germ cells vs somatic cells, the presence of a uH2A body beginning in early spermatocytes, and high levels of uH2A on the Y chromosome and little or none on the X. The former is intriguing because this modification is usually associated with silencing, yet the Y chromosome is active in spermatogenic cells. The authors interpret some of their data as implying a lack of dosage compensation of the X chromosome in spermatocytes.

The data are believable and new, but it is not fully clear how to interpret them. The paper's interpretations rely on subtractive logic to parse results from mixtures of cells down to cell type, extracting spermatogonia, spermatocyte, etc. features by comparing bam mutants (only spermatogonia) to aly mutants (spermatogonia and early spermatocytes but no later stages) to wildtype (all spermatogenic stages), and extracting testis germline data by comparison to wing disc soma; their FACS sorted spermatocytes also have heterogeneity. I recognize that the present paper was a lot of work and am not suggesting that the authors redo their study using methods that give more purity and precision of stage (https://doi.org/10.1126/science.aal3096, https://doi.org/10.1101/gad.335331.119), but they should be aware of them and of their results.

The conclusions about dosage compensation are indirect, but are consistent with the current model documented in the studies cited by the authors, as well as earlier studies (doi: 10.1186/jbiol30).