Analyzing Drosophila female germline chromatin

A. Stages of female gamete development in an ovariole. A single germline stem cell (GSC) and germline cyst are shown. To the right, a germarium is illustrated showing regions 1-3. Follicles in an ovariole are pictured at a lower magnification starting with stage 2 (S2). Below the diagrams, major events are summarized. B. FACS purification (upper panel) of 4c-512c germ cells for ATAC after earlier separation of 2c and 4c GSCs, and 2c-16c follicle cells (see Figure S2). DAPI, DNA content; Inset shows further purification based on germ cell marker expression (tomato). Below, follicle stages collected for RNAseq and Chip-seq are show by black bars. C. The number of genes expressed (>1 tpm, blue) and off (<1 tpm, orange) in GSCs decreases as germ cell development proceeds downstream. D. Box plot showing a decline in total ATAC peaks from GSCs to young ovaries (before S6) to whole ovaries (S6-9). E. Stem cells already express a high fraction of 79 oocyte maternal effect genes defined in Drosophila genetic screens.

Heterochromatin forms in cysts downstream from the GSC

A,B. Germaria stained for (A) H3K9me3-binding protein HP1a (pink) and CENP-A (green), or (B) H3K9me3 (green), fusome antibody 1B1 (pink) and DAPI (blue). C. H3K9me3 Chip-seq of unambiguously mapped reads spanning chromosome 2 (lower) and 3 (upper) in 250 kb bins from GSC, 4C NC or 8C NC. D. Plots showing relative enrichment of reads mapping to TEs or to satellite sequences in 4C, and 8C germ cells relative to GSCs. E. A representative euchromatic hsGFP insertion after heat shock expresses GFP (green) in GSCs and nearly all downstream cyst and follicular NC (arrows). In contrast a typical heterochromatic insertion after heat shock only expresses in GSCs and early cysts (upper arrow), but has become repressed in 16-cell cysts and NC in meiotic and later follicles (lower arrow; other labeled cells are somatic). F. Repression of heterochromatic hsGFP expression after heat shock (+hs) (Control) requires the H3K9 methylase eggless/SETDB1 (eggRNAi). G. Diagram shows the ratio of hsGFP expression in piwi(GLKD) or control genetic backgrounds from individual hsGFP insertions (points) at the indicated developmental stages (Figure 1A) colored based on insert location in euchromatin (pink), pericentric chromatin (green) or centric heterochromatin (blue). note: SC = GSC; CB = cystoblast (1st cell of germline cyst). H. Summary diagram of Drosophila female germ cell generational cycle showing two highly potent stem cell chromatin states (pink) beginning with the GSC or zygote and their downstream developmental trajectories. Both the GSC (maternal) and zygotic trajectories quickly undergo heterochromatin formation (blue) and then somatic cell production dependent on Polycomb repression (green). see text and Figure S2 for further details).

E(z)-dependent repression contributes to sex-differential gene expression

A. Diagram of Drosophila PRC1 and PRC2. B. Chromatin changes around the GSC differentiation gene bgcn. The promoter-associated H3K27ac peak is lost by 32cNCs, when H3K27me3 has begun to accumulate. C. The number of genes strongly downregulated by E(z) in young ovaries and their predominant tissue of expression. D. Chromatin changes around the CG43088 testis gene related to a transposon. E. The male GSC gene escargot (esg) downregulated by OVS6 shows increased H3K27me3. F. The chinmo gene encoding a transcription regulator required in male and female germ cells is repressed as in E. G. The testis regulatory gene aly, is repressed as in E. H. The tdrdl5 male germ cell regulator is repressed as in C. I. Phf7 gene regulation. J. Summary of E(z)-dependent repression of male and somatic genes during early female gene expression. K. Three sub classes of genes downregulated by E(z).

Maintenance and exit from the GSC state

A. Ovarioles subjected to white gene (wGLKD) or nejire gene (nejireGLKD) germline knockdown. Levels of H3K27ac (green) are greatly reduced in nejGLKD and germ cell development is arrested at stage 1-2. Hts staining (1B1, purple) highlights cell membranes and fusomes. B. Chromatin tracks around the CGG gene piwi which is expressed throughout oogenesis. H3K27me3 does not increase. C. mRNA abundance vs the transcription start site (TSS) levels of H3K27ac are plotted versus GSC gene expression. Genes are colored according to hidden Markov model (HMM) calls of H3K27me3 levels. D. The number of gene-associated H3K27ac peaks (upper) or ATAC peaks (lower) in GSCs is plotted versus log10 peak score. 81% of H3K27ac peaks and 68% of ATAC peaks are associated with TSSs; the remaining peaks may be associated with enhancer regions. E. Examples GSC expressed genes that are downregulated in young ovaries in a E(z)-dependent manner (Table S2). The H3K27ac patterns of two such genes, blanks and eIF4E3 are shown. F.Germarisum immunostained for H3K29ac showing low levels in early germ cells (dashed pink circle) and increase in S1-S3 nurse cells (purple dashed circles). G. Plot of GSC mRNA abundance vs expression change in young ovary compared to GSCs. A strong correlation can be seen between genes with changes in mRNA levels and changes in H3K27ac levels. Genes (dots) are colored according to changes in ac = H3K27ac levels.

Genes induced in germ cells downstream from GSCs mediate a metabolic and growth transition

A. Germ cell-expressed transcription regulators with the largest fold change increases in young NC (OVS6) compared to GSCs. B. GO terms for genes upregulated in young ovaries vs GSCs. The GO categories reveal that a dramatic metabolic shift occurs between GSCs and OVS6 nurse cells. C. Expression values (tpm) in GSCs and OVS6 NCs of glycolytic genes showing mostly upregulation but a shutoff of Ldh. D. Diagram showing upregulated transmembrane transport genes (1st GO category in B). Sub-cellular localization of a sample (20) of membrane transporters (Dros name/Mouse name) is indicated (arrows) on the cell drawing showing plasma membrane (red), mitochondrion (blue) or Golgi (orange). along with and their fold change (increase) in NC. Many are key regulators of cellular import or mitochondrial metabolism that help generate the precursor nucleotides, lipids and carbohydrates needed for rapid cellular growth. For complete list (Table S5).

The GSC state provides high immunity to transposable elements (TEs)

A. Proposed amplification of TE resistance by a germline cyst or syncytial stage downstream from a highly potent stem cell by amplifying and sharing piRNA (blue lines) between all connected cells following TE1 movement in one cell. B. Summed expression (tpm) of conserved germline genes (CGG) genes (blue) involved in transposon regulation and nuclear piwi genes (orange). C. Illustrative diagram of cluster analysis in a male GSC lineage with two cyst divisions and a single meiotic division leading to 8 sperm. The number of sperm with identical TE insertions depends of the time of insertion during cyst formation, as shown by 3 different TE examples that insert at different stages. D. Model of P element (triangles) copy number increase on chromosomes (horizontal lines) during a cyst cell or meiotic germ cell cycle by “replication timing”. red = ORC proteins, Green = MCM proteins, black line shows postulated repression prior to loss of pre-replication complex in S phase by origin activation or fork passage. blue arrow shown transposition from a replicated to an unreplicated region (recognized by unfired pre-replication complex during later S phase (modified from Spradling et al. 2011). E. Like D, but in a GSC whose short G1 is proposed activate all origins simultaneously.

Growth profiles (total germ cell volume) of Drosophila ovarian follicles

A. Growth of wild type follicles shown by plotting volume (log10) determined as described in Methods vs integrated DAPI (log10). An example of follicles with GFP labeled germ cells is shown at the right. Stages of the follicles are indicated by color key on the right. B. Similar methods applied to reveal the growth of control vs yolkless follicles. C. Similar methods used to determine the growth of control and Cap/Df follicles.

The Drosophila female germ cell cycle.

A. The Drosophila female germ cell generational cycle. Starting with adult germline stem cells (GSCs, magenta, upper right) germ cells form germline cysts (tan) that move through germarium regions 1-3 and bud off as stage 2 follicles (S2). Mature S14 oocytes are fertilized and undergo 14 preblastoderm syncytial divisions, with pole cells budding off at division 9 to form primordial germ cells (PGCs). About 10 PGCs complete migration to the embryonic ovary, that develops new germaria prior to adulthood. The full cycle entails 19-20 germ cell divisions entailing an estimated >10,000-fold cytoplasmic volume increase.

Isolation of GSC and NC nuclei by FACs sorting.

A. The Drosophila female germ cell generational cycle. Starting with adult germline stem cells (GSCs, magenta, upper right) germ cells form germline cysts (tan) that move through germarium regions 1-3 and bud off as stage 2 follicles (S2). Mature S14 oocytes are fertilized and undergo 14 preblastoderm syncytial divisions, with pole cells budding off at division 9 to form primordial germ cells (PGCs). About 10 PGCs complete migration to the embryonic ovary, that develops new germaria prior to adulthood. The full cycle entails 19-20 germ cell divisions entailing an estimated >10,000-fold cytoplasmic volume increase.

Genes with high H3K27ac levels in GSCs cluster in heterochromatin and two

A. Diagram of the Drosophila chromosomes showing zones of clustered genes with high H3K27ac that are expressed in GSCs and downstream germ cells. heterochromatin = blue boxes. numbers below line are coordinates in Mb. numbers above line are cytogenetic regions.

Analysis of chromatin changes at enhancers of Myc during oogenesis

A. myc region chromatin accessibility assayed by ATACseq in GSCs and 4C-32C nurse cells (NC). B. Genomic DNA fragments from this region (1-11, indicated) cloned and integrated within Janelia GAL4 lines (Jennet et al. 2012) are shown. C. A summary of the GFP expression driven by 8 tested clones fragments. D. Enlarged view of GFP expression from clone 8 (R10G10), which is similar to clone 7 and is shown below. The pattern of expression is indicated to show low level (clones 7 and 8) and higher level (clones 9 and 10) GFP expression starting about stage 4.