Volume 42, Issue 6, Pages 837-844 (June 2011) The Polycomb Group Mutant esc Leads to Augmented Levels of Paused Pol II in the Drosophila Embryo  Vivek S. Chopra, David A. Hendrix, Leighton J. Core, Chiahao Tsui, John T. Lis, Michael Levine  Molecular Cell  Volume 42, Issue 6, Pages 837-844 (June 2011) DOI: 10.1016/j.molcel.2011.05.009 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 42, 837-844DOI: (10.1016/j.molcel.2011.05.009) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Augmented Pol II Occupancy in esc Mutant Embryos (A) The three different genetic backgrounds used for ChIP-Seq assays. Wild-type (WT) embryos have normal function of PRC2 and Abd-B. There is diminished PRC2 function in esc mutant, leading to ectopic expression of Abd-B and the conversion of all embryonic segments to an eighth abdominal segment (A8) morphology. Abd-B was overexpressed in WT embryos via heat shock using hs-GAL4 and UAS-Abd-B transgenes (also see Supplemental Experimental Procedures). (B) Pol II ChIP-Seq assays in early (0–2 hr) and late (18–24 hr) WT embryos. They contain overlapping subsets of stalled genes (432 early, 538 late, and 1231 overlap). These genes contain a stalling index of at least 10 (see Figure S1). Each red mark represents a single gene. Marks located within the blue (18–24 hr) or yellow (0–2 hr) shaded regions correspond to genes that are stalled/paused in just one of the genotypes. The red marks located within nonshaded regions of the plot are stalled/paused in both genotypes. (C) Pol II stalling/pausing in late (18–24 hr) WT and esc mutant embryos. There are ∼3500 stalled genes in esc mutants, including 2133 genes which show “ectopic stalling” only in mutant embryos (class II genes). As in (B), red marks falling within the shaded regions (green, esc; blue, WT) represent genes that are specifically stalled in only one of the genotypes. (D) Relative levels of Pol II ChIP-Seq binding in WT and esc embryos for class II genes (stalled/paused in esc mutants, but not WT embryos). esc mutant embryos at 18–24 hr exhibit a 6.5-fold increase over background levels (green). A 3.5-fold (blue) increase was observed for 18–24 hr WT embryos. Just 3-fold (pink) and 2-fold (red) increases were observed for 18–24 hr embryos with or without heat shock Abd-B, respectively. (E) The stalled and inactive (“off”) genes seen in 18–24 hr esc embryos were divided into three subsets: those showing 1- to 2-fold (blue), 2- to 3-fold (green), and greater than a 3-fold (red) increase in Pol II stalling as compared with WT embryos (see Figure 3A). Genes in the greater than 3-fold category (1485/2133 [70%] of class II genes) are enriched for putative targets of PRC2, including Hox genes and head patterning genes (e.g., orthodenticle and aristaless; see Figure S2A). All three subsets of stalled genes also exhibit strong GRO-Seq signals, confirming that they are “paused” and capable of productive elongation. Molecular Cell 2011 42, 837-844DOI: (10.1016/j.molcel.2011.05.009) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Dynamics of Pol II Stalling/Pausing in WT and esc Embryos (A) The Sex combs reduced (Scr) gene is not paused in early (0–2 hr) (track 2), mid (8–12 hr) (track 3), and late (18–24 hr) (track 4) WT embryos. Scr is a likely target of PRC2 regulation, and H3K27me3 signals are observed in early (0–2 hr) WT embryos (track 1). Enhanced Pol II occupancy is observed at both Scr promoters in midstage (8–12 hr) (track 5) and late (18–24 hr) (track 6) esc mutant embryos (black arrows). In contrast, no increase in Pol II stalling is observed in WT embryos containing ectopic expression of Abd-B via heat shock (track 7). GRO-Seq assays indicate that the Scr promoters contain elongation-competent (paused) Pol II (tracks 8 and 9). (B) The toe-eyg complex was analyzed for Pol II occupancy and H3K27me3 modification. The toe and eyg genes are not stalled in early (0–2 hr) (track 2) and mid (8–12 hr) (track 3) stage embryos, but exhibit some Pol II pausing (for eyg promoter) in late (18–24 hr) (track 4) WT embryos. These genes are likely targets of PRC2 regulation, since H3K27me3 signals are distributed throughout the complex in early (0–2 hr) WT embryos (track 1). In midstage (8–12 hr) (track 5) esc mutant embryos, we see augmented Pol II stalling at the eyg promoter (black arrow) and both toe and eyg exhibit stalling in older (18–24 hr) (track 6) esc mutant embryos. GRO-Seq assays suggest that Pol II is paused at both promoters (arrows, tracks 8 and 9). There is no significant Pol II stalling of toe and eyg promoters in WT embryos (track 4), nor in embryos containing ectopic Abd-B expression via heat shock (track 7). Note that 0–2 hr embryos are likely to be biased toward 2 hr (or even older) due to the geometric increase in the number of nuclei during precellular development. Molecular Cell 2011 42, 837-844DOI: (10.1016/j.molcel.2011.05.009) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Transcriptional Profiling of WT and esc Mutant Embryos (A) The stalled genes that are inactive in esc mutants were divided into three subsets (similar to Figure 1D): 1- to 2-fold (blue), 2- to 3-fold (green), and more than a 3-fold (red) increase in Pol II stalling in esc as compared with WT embryos. (B) The same three subsets of genes exhibit increased H3K4me3 binding (a trxG mark) when normalized to WT embryos. This modification is most enriched ∼200–300 bp downstream of the +1 transcription start site. (C) H3K27me3 ChIP-Seq read counts at stalled and nonstalled promoters in 18–24 hr WT embryos. There is a slight diminishment of H3K27me3 read counts at the promoter regions of stalled genes (red and blue lines) as compared with nonstalled genes (yellow and green lines). (D) H3K27me3 ChIP-Seq read counts at stalled and nonstalled promoters in esc mutants. There is a significant depletion of H3K27me3 read counts as compared with WT embryos (Figure 3C). There are also diminished read counts in the promoter regions of active nonstalled genes (green line) as compared with inactive nonstalled genes (yellow line). (E) Analysis of the ∼1500 “top quartile” genes (Gilchrist et al., 2010). Relative changes in Pol II (red), H3K4me3 (green), and H3K27me3 levels in esc versus WT embryos. There is a ∼1.8-fold increase in Pol II, 3.5-fold increase in H3K4me3, and reduced levels of H3K27me3 in esc embryos as compared with WT embryos. (F) PHO-regulated genes (∼500 genes) (Oktaba et al., 2008). Relative changes in Pol II binding in esc mutants and WT embryos, as compared with background controls. There is a 9-fold (red) increase in Pol II binding in esc mutants and a 5-fold (green) increase in WT embryos (also see Table S4 and Figure S3F). Molecular Cell 2011 42, 837-844DOI: (10.1016/j.molcel.2011.05.009) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Binding Threshold Model for Paused Pol II (A) Core promoter pausing elements in different classes of stalled genes. Nearly 36% of class I genes (Pol II stalling in both WT and esc embryos) contain both an initiator element at the transcription start site (Inr) and a pause button (PB) motif located within 50 bp downstream of the start site, most often at approximately +30 bp. Only ∼16% of class I genes lack both promoter elements. A flip-flop correlation was observed for class II genes (Pol II stalling only in esc mutants). Only ∼18% contain both Inr and PB elements, while ∼34% of all class II genes lack both elements. Thus, there is a 2-fold enrichment in the occurrence of Inr and PB elements in class I versus class II genes. (B) Threshold model for paused Pol II. In wild-type embryos, Pol II (light blue) overcomes repression by PRC2 due to the occurrence of core promoter pausing elements, such as the Inr and DPE/PB (the PB motif is a GC-rich variant of the DPE, which is located from +28 to +32 bp downstream of the +1 transcription start site). H3K4me3 (light green nucleosome) is detected just downstream of stalled Pol II. In contrast, class II genes contain H3K27me3 at the core promoter and contain reduced levels of H3K4me3 in the downstream nucleosome. In esc mutants, diminished PRC2 activity leads to enhanced Pol II binding at class I promoters (dark blue) and reduced H3K27me3 at the promoter regions of class II genes. Pol II is now able to bind to class II promoters due to diminished levels of repressive nucleosomes. According to this model, the switch in paused Pol II is influenced by the presence or absence of promoter pausing elements and the action of PcG repressive complexes. The reduction in PRC2 activity in esc mutants lowers the threshold of Pol II binding, even for genes lacking core promoter pausing elements. Molecular Cell 2011 42, 837-844DOI: (10.1016/j.molcel.2011.05.009) Copyright © 2011 Elsevier Inc. Terms and Conditions