H3K4 erasure in mes-4 primordial germ cells (PGCs) is normal, but exhibits precocious reappearance. H3K4me2 staining in (A-C) early and (B-D) late stage primordial germ cells (PGCs of embryos from (A,C) wild type, (B,D) mes-4(bn85) and (C,E) mes-2 mothers. DAPI, red; H5, green; PGL-1, blue in merged images. In the lower panels, PGCs (boxed; arrows) are enlarged, and the H3K4me2 staining is shown in grayscale (left). Arrows mark PGCs.

H3K36me3 ChIP-seq in met-1 embryos detects maternal effect sterile (MES)-4-mediated histone methylation. MES-4-mediated H3K36me3 (green), pan-histone H3 (brown) and input (gray) profiles on an autosome (chromosome IV) and the X chromosome in met-1 mutant late embryos. The red circle indicates MES-4-mediated H3K36me3 signal on the left tip of the X chromosome. The signals for pan-H3 and input are partly magnified relative to that of H3K36me3. Averages from two independent experiments are shown.

H3K36 methylation by MES-4 is unaffected by Pol II knockdown in adult germ cells. Wild type and met-1(n4337) animals soaked and fed ama-1 double-stranded (ds)RNAi for >50 hours and whole-mount fixed gonads were stained for Pol II (AMA-1; left columns) and H3K36me3 (left middle) or H3K4me3 (right middle). Comparable pachytene stage nuclei of indicated genotypes and treatment are shown. Note that both Pol II and H3K4me3 signals were significantly reduced ama-1 RNAi, whereas H3K36me3 was unaffected.

Pol II CTD phosphorylation is uniquely regulated in primordial germ cells (PGCs). (A) H5 staining in ama-1 (RNAi) is depleted by ama-1(RNAi). (B) cdk-9 (RNAi) embryo ablates H5 staining in all cells except the PGCs. (C) tlk-1 (RNAi) ablates H5 staining in many somatic cells, but the PGCs are often resistant. (D) cdk-9; tlk-1(RNAi) consistently ablates all H5 staining, including that in PGCs. Note that polyploid nuclei are observed in the tlk-1 (RNAi) embryo, as described previously [71]. H5 and DAPI staining (middle and right, respectively) are shown in grayscale with the merged image (left). Arrows indicate the PGCs.

MES-4 is unable to provide detectable de novo H3K36 methylation. The maternal effect sterile (MES)-4 protein is expressed zygotically in the primordial germ cells (PGCs) of (A,B) embryos or (C,D) L1 larvae could not provide detectable H3K36me3 to animals that did not inherit maternal chromatin with H3K36me3. H3K36me3 was detectable in (A) mes-4 M-Z+;met-1 M-Z+ larval PGCs, but not in (B) mes-4 M-Z+;met-1 M-Z- PGCs. Similarly H3K36me3 was observed in (C), mes-4 M-Z+;met-1 M-Z+ ~500 cell embryo PGCs but not in (D) mes-4 M-Z+;met-1 M-Z- PGCs. MES-4 indicates anti-MES-4 antibody staining. Arrows mark PGCs. Scale bars: 3 μm.

A hypophosphorylated form of Pol II remains associated with the autosomes, but not the X chromosomes, in late primordial germ cells (PGCs). (A) Pol II is still on DNA late PGCs. DAPI, red; Pol II (8WG16 antibody) staining, green; PGL-1 staining, blue. Lower panels show enlarged PGCs and the separated channel for Pol II staining in grayscale. Arrows point to regions in the PGCs that lack Pol II staining. (B) The regions of Pol II exclusion overlap with regions lacking MES-4, which specifically associates with the autosomes at all stages that have been examined. Arrows point to the region of X chromosomes. Scale bars: (A) 10 μm; (B) 3 μm.

The maternal effect sterile (MES)-4 protein marks genes expressed in germ cells but not soma. (A-E) Scatter plot comparison between H3K36me3 in met-1 (mediated by MES-4) and H3K36me3 in wild type (mediated by MES-4 and methyltransferase (MET)-1). Gray dots show the log₁₀ average read counts for base pairs between the transcription start and end site for all genes. Red dots highlight (A) X-linked genes, (B) germline-enriched genes, (C) ubiquitously expressed genes, (D) soma-expressed genes and (E) larval/adult germline-specific genes. See text and supplemental experimental procedures for descriptions of gene sets.

H3K36 methylation dynamics during embryogenesis. (A) MES-4 is required for the maintenance of H3K36me3 during early embryonic cell divisions. H3K36me3 (left) and DAPI staining (right) in mes-4 (bn85) M-Z- (top panels) and wild type (bottom panels) are shown in grayscale. Note that in mes-4 embryos, H3K36me3 that is prominently observed in dividing 1-cell stage embryo (embryo a), becomes barely detectable by the 2 to 4 cell stage (embryo b) and almost undetectable at the 6 to 8 cell stage (embryo c). Arrows indicate polar bodies. (B) H3K36me3 staining in wild type (top row), met-1(n4337) (middle row) and mes-4(bn85) (bottom row) at different embryonic stages (~50, ~100 and ~550 cells, as indicated on the top). DAPI, red; H5, green; PGL-1, blue in merged images. (C) H3K36me3 staining is not detectable in mes-4(bn73); met-1(n3347) double mutant, Arrows indicate primordial germ cells (PGCs.

H3K36 methylation by maternal effect sterile (MES)-4 is enriched in transcribed regions of germline expressed loci. (A) Screen shots of H3K36me3 (green), panH3 (brown) and input patterns (gray) in wild type and met-1 background mapped onto the UCSC genome browser for several regions are shown together with the annotated gene models (blue, top of each panel). (Top) MES-4-mediated H3K36me3 was enriched on germline-expressed genes, gst-1 and nos-1(red box), but not on a somatic gene ptp-3 (blue box), compared with the methylation pattern in wild type, whereas the methylation pattern on a ubiquitously expressed gene let-858 (green box) was similar in both wild type and met-1 backgrounds. (Bottom left): daz-1, which is essential for meiotic progression during oogenesis and is expressed specifically in the larval/adult germline [72] is a locus at which MES-4-mediated methylation is significantly enriched. (Bottom right): Another example of specific enrichment of MES-4-mediated H3K36me3 on the adult germline-specific gene oma-1, which is required for oocyte maturation. The y-axis is the signal intensity (the number of illumina GA sequence reads). (B) Gene profiles showing MES-4-mediated H3K36me3 on larval/adult germline-specific genes (red; see also Figure 10E) and soma-expressed genes (blue; see also Figure 10D) in met-1 embryos. Values are average scores for 50 bp intervals in the 2.5 kb surrounding the transcription start site (TSS) and the transcription end site (TES). Error bars indicate the 95% confidence interval.

Unique and dynamic regulation of Pol II in the embryonic germline. (A) Transcription status and histone modification dynamics in C. elegans early embryonic blastomeres. The asymmetric divisions of P-lineage cells (P0 to P3) produce both germline and somatic blastomeres. The last P cell division (P4) is symmetric and produces two germline-committed primordial germ cells (PGCs), Z2 and Z3 and these cells arrest at G2 phase through the rest of embryogenesis [68]. Two maternally expressed CCCH zinc finger proteins, OMA-1/2 and PIE-1, act sequentially in P0 to P4 to maintain transcriptional quiescence independently of the chromatin environment [14,69,70] (orange line). At the P4 division, PIE-1 is degraded quickly and 'active Pol II' (H5 staining; magenta line) appears in the PGCs (circled by blue dotted line). H3K36me3 (black) and H3K4me2 (green) are initially present in both the transcriptionally quiescent P-lineage and their somatic sisters, where transcription is activated. MES-4, which is essential for germ cell viability, produces H3K36me in the germline precursors independently of transcription [21]. H3K4me2 is also maintained through the P-lineage by an unidentified mechanism, but the level of H3K4me2 becomes almost undetectable in the PGCs [15] (indicated by dotted green lines). (B-L) H5 staining of Pol II Ser2P in the PGCs of (B-E) wild type, (F-I) mes-4 and (J-L) mes-2 during embryogenesis; (B,F)~150 minutes post-fertilization at 22°C, ~26-cell stage; (C,G,J) ~200 minutes, ~90-cell stage; (D,H,K) ~450 minutes, ~1.5-fold stage; (E,I,L) >500 minutes, 2- to 3-fold stages. PGCs are boxed and shown by arrows. DAPI staining, red; H5 antibody staining, green; PGL-1 (germline marker) staining, blue. In the lower panels, PGCs are enlarged, and the separated channels for DAPI and H5 staining are shown in grayscale. Scale bars: 10 μm.