Profiling of the histone H3K4me2 modification in the Drosophila testis.

(A) Schematic of male germline stages in Drosophila. Germline stem cells (GSCs, fuschia) are located in the apical tip of the testis. After an asymmetric division a progeny spermatogonium (purple) undergoes 4 rounds of mitotic divisions. After one last S phase cells grow over ∼3 days as spermatocytes (blue, light green, dark green) before meiosis (red). Post-meiotic differentiation produces mature sperm (black) with elongated nuclei. Somatic cell types of the testis are not shown.

(B-E) Distribution of the H3K4me2 modification in testes from bam mutants, from aly mutants, from wildtype animals, and from wing imaginal discs.

(B) H3K4me2 around the GSC-expressed nanos gene. Neighboring genes show peaks in all samples, while low signal across nanos is highest in testes from bam mutants, and apparent in all three testes samples.

(C) H3K4me2 around the spermatocyte-expressed loopin-1 gene. H3K4me2 signal appears in aly mutant samples (which contain early spermatocytes) and reach high levels in wildtype testes (which include later stages of spermatogenesis).

(D) H3K4me2 around the meiotically-expressed genes Mst36Fa and Mst36Fb genes. Signal across these genes only appears in wildtype testes.

(E) H3K4me2 around the abd-A and Abd-B genes, which are expressed in somatic cells of the testis.

Changes in the histone H3K4me2 modification in germline-expressed genes.

H3K4me2 signal around gene promoters (-200–+500 bp) with transcripts enriched in specific germline cell types. Transcript expression was derived from FCA snRNA-seq clustering [Raz et al, 2023].

(A) Expression (snRNA-seq) in wildtype testes and H3K4me2 enrichment in bam mutant, in aly mutant, and in wildtype testes in germline stages. Z-scores for H3K4me2 signal were calculated between the three genotypes. Notable germline-enriched genes are indicated, including those expressed in GSCs, in meiosis, or for cell division (black), linked to the Y chromosome (purple), or expressed post-meiotically (orange).

(B) Selected examples of promoters with germline-enriched expression. Expression z-scores from FCA snRNA-seq across 18 germline clusters (Raz et al. 2023) and H3K4me2 enrichment in bam mutant, in aly mutant, and in wildtype testes.

(C) Distribution of H3K4me2 around promoters with germline-enriched expression. The testis somatic category comprises the top tercile of promoters with somatic cell-type expression in snRNA-seq data (Raz et al. 2023). Only promoters with no promoter of a second gene within 1 kb upstream are shown.

Profiling RNA Polymerase II in isolated spermatocytes.

(A) An adult testis carrying a UASRFP construct induced by a bamGAL4 driver and a hephGFP construct. Gonial cells are labeled red while spermatocytes are labeled green with fluorescent proteins.

(B) FACS plots of recorded events from dissociated testes for forward scatter (FSC) and GFP signal. Boxes indicate events collected for chromatin profiling.

(C) Distribution of RNAPIIS2p at the spermatocyte-expressed betaTub85D gene in isolated spermatocytes and in wing imaginal discs.

(D) Distribution of RNAPIIS2p at the broadly-expressed stc gene in isolated spermatocytes and in wing imaginal discs. RNAPIIS2p is strongly localized at the stc promoter in wing imaginal discs, but more evenly distributed in spermatocytes.

(E) Selected examples of genes with late germline expression and for protamines. Expression z- scores from FCA snRNA-seq across 18 germline clusters (Raz et al., 2023) and RNAPIIS2p enrichment in isolated spermatocytes.

(F) Enrichment of RNAPIIS2p in isolated spermatocytes and in wing imaginal discs across genes with germline-enriched transcripts.

Chromosomal distribution of RNA polymerase II and H4K16ac in isolated spermatocytes.

(A) CIRCOS plot of RNAPIIS2p across Drosophila chromosomes. The signal (black) in IgG controls, in wing imaginal discs, and in isolated spermatocytes is shown in internal rings, and the log2 fold-change of signals between spermatocytes and wings is shown in the outer ring.

(B) Enrichment of RNAPIIs2p across gene bodies in wing imaginal discs, in whole dissociated testes, and in isolated spermatocytes separated by chromosomal location. Scores are scaled to the median score on the 2nd and 3rd chromosomes.

(C) CIRCOS plot of the dosage-compensation marker histone H4K16ac across Drosophila chromosomes. Signal (black) in IgG controls, in wing imaginal discs, and in isolated spermatocytes is shown in internal rings, and the log2 fold-change of signals between spermatocytes and wings is shown in the outer ring.

Chromosomal distribution of repressive histone modifications in isolated spermatocytes.

CIRCOS plots across Drosophila chromosomes show signal (black) in IgG controls, in wing imaginal discs, and in isolated spermatocytes in internal rings, and the log2 fold- change of signals between spermatocytes and wings in the outer ring.

(A) Distribution of the heterochromatin-silencing marker H3K9me2.

(B) Distribution of the Polycomb-silencing marker H3K27me3.

Chromosomal distribution of ubiquitinylated histone H2A in isolated spermatocytes.

(A) CIRCOS plots of uH2A across Drosophila chromosomes in IgG controls, in wing imaginal discs, and in isolated spermatocytes.

(B) Immunostaining of uHA (blue) and the nucleolar marker fibrillarin (red) on germline nuclei. Early-, mid-, and late-spermatocyte stage were identified by PcGFP (green) localization pattern in wildtype spermatocytes and in X/O spermatocytes. Testes from bam mutants contain gonial cells, while testes from aly mutants contain mostly early spermatocytes.

(C) Correspondence of uH2A and RNAPIIS2p signals around the promoters of genes on the Y chromosome in wing imaginal discs and in isolated spermatocytes.

(D) Correspondence of uH2A and RNAPIIS2p signals across transposon consensus sequences in wing imaginal discs and in isolated spermatocytes.