Cohesin selectively binds and regulates genes with paused RNA polymerase

Abstract

Background: The cohesin complex mediates sister chromatid cohesion and regulates gene transcription. Prior studies show that cohesin preferentially binds and regulates genes that control growth and differentiation and that even mild disruption of cohesin function alters development. Here we investigate how cohesin specifically recognizes and regulates genes that control development in Drosophila.

Results: Genome-wide analyses show that cohesin selectively binds genes in which RNA polymerase II (Pol II) pauses just downstream of the transcription start site. These genes often have GAGA factor (GAF) binding sites 100 base pairs (bp) upstream of the start site, and GT dinucleotide repeats 50 to 800 bp downstream in the plus strand. They have low levels of histone H3 lysine 36 trimethylation (H3K36me3) associated with transcriptional elongation, even when highly transcribed. Cohesin depletion does not reduce polymerase pausing, in contrast to depletion of the NELF (negative elongation factor) pausing complex. Cohesin, NELF, and Spt5 pausing and elongation factor knockdown experiments indicate that cohesin does not inhibit binding of polymerase to promoters or physically block transcriptional elongation, but at genes that it strongly represses, it hinders transition of paused polymerase to elongation at a step distinct from those controlled by Spt5 and NELF.

Conclusions: Our findings argue that cohesin and pausing factors are recruited independently to the same genes, perhaps by GAF and the GT repeats, and that their combined action determines the level of actively elongating RNA polymerase.

Figure 1

Enrichment of GAF Binding Sites and GT Repeats in Cohesin-Binding Genes (A) Combined frequency of GAGAG and CTCTC, binding sequences for GAGA factor (GAF), at positions from −1 kb to +1 kb around the TSS of 506 cohesin-binding genes (blue) and 1040 genes that do not bind cohesin (green). (B) GAGAG and CTCTC frequency increases with the stringency with which cohesin binding is called (blue, p ≤ 10⁻¹⁵; green, p ≤ 10⁻⁹; gray, p ≤ 10⁻³). (C) Frequency of GTGTG from −1 kb to +1 kb in cohesin-binding (blue) and non-binding (green) genes. (D) Frequency of GTGTG (blue) and CACAC (green) in cohesin-binding genes. (E) Frequency of GTGTG in non-coding (blue) and coding (green) sequences of cohesin-binding genes. (F) Frequency of a GT or TG dinucleotide sequence following another in the 506 cohesin-binding (blue) and 1040 non-binding genes (yellow-orange) in the 1 kb region downstream of the TSS. The genes are in order of decreasing consecutive dinucleotide frequency. Comparison of genome-wide binding of Nipped-B to GAGA factor (GAF) in Figure S1 indicates that the GAGAG sequences enriched in cohesin-binding genes are functional binding sites.

Figure 2

Cohesin Selectively Binds Genes with Paused Polymerase but Cohesin Depletion Does Not Reduce Pausing (A) Average abundance of 3′ ends of short RNA transcripts, a marker of paused polymerase [33] in cohesin-binding (blue) and non-binding (green) genes at the transcription start sites. (B) Western blots showing RNAi depletion of NELF-B, NELF-E, and GAF in BG3 cells. (C) Effects of NELF, GAF, Nipped-B and Rad21 depletion on polymerase pausing at the path, rho, dm, HLHm3 and invected genes as measured by KMnO₄ footprinting. G/A lanes contain products from BG3 genomic DNA from the Maxam-Gilbert G/A reaction, and DNA lanes contain products from KMnO₄-treated purified genomic DNA. The remaining lanes contain products produced from genomic DNA isolated from KMnO₄-treated BG3 cells after the indicated RNAi treatments.

Figure 3

Cohesin Does Not Interfere with Transcriptional Induction or Elongation at EcR Gene in BG3 Cells (A) Map of EcR and association of PolII, combined Smc1-Nipped-B, GAF, and the H3K36me1 and H3K36me3 histone modifications determined by ChIP-chip in BG3 cells [15, 29, 30]. Bars underneath the ChIP profiles indicate where binding is called at p ≤ 10⁻³. Positions of RT-PCR probes are indicated in blue. (B) Levels of transcripts containing p1, p2, p3 and 3′ exon sequences before (blue) and after (red) ecdysone induction for 60 min. Cells were untreated (Mock) or pretreated with the indicated RNAi (Rad21, Nipped-B, NELF-B, GAF) for 4 to 6 days. Error bars are standard errors, calculated using all RT-PCR replicates in three independent experiments. (C) Time courses of ecdysone induction showing the relative increases in transcripts containing the indicated probe sequences, with or without Rad21 depletion. Time courses after Nipped-B, GAF and NELF-B knockdown are in Figure S2. The curves shown are third order polynomial fits. Some replicates included 45 min time points (not shown) showing that intron B and C RNA reach peak levels around 45 min and then decrease. Error bars are standard errors, calculated using all RT-PCR replicates from three independent experiments.

Figure 4

Effects of Cohesin (Rad21), Nipped-B, Spt5 and NELF-B Depletion on big bang (bbg) Transcripts and PolII binding in BG3 Cells (A) Map of bbg with ChIP-chip profiles. PolII ChIP identified p2 (unannotated) and p3 as the active promoters. RT-PCR probes are indicated in blue. (B) Western blots showing typical RNAi depletion of Rad21 and Spt5. (C) Changes in levels of transcripts containing the indicated probe sequences relative to control (Mock) after five days of the indicated RNAi treatments. Mock control p2 transcripts are ~0.6% the level of RpL32 transcripts, p3 are ~0.15%, and 3′ exon transcripts are ~0.22%. Error bars are standard errors, calculated using all RT-PCR replications from two independent experiments. Other times of RNAi treatment and other independent experiments are shown in Figure S4. (D) Enrichment of the indicated probe sequences relative to an intergenic region (see Methods) after ChIP using anti-Rpb3 and anti-Ser2P PolII antibodies five days after RNAi treatment. Error bars are standard errors calculated using all RT-PCR replicates from ChIP of two to three independent chromatin preparations. ChIP for Rad21 and Nipped-B binding after Rad21 and Spt5 depletion are shown in Figure S3.

Figure 5

Effects of Rad21, NELF-B and Spt5 Depletion on Transcripts of terribly reduced optic lobes (trol) and PolII Binding in BG3 Cells (A) Map of trol, with ChIP-chip data and locations of RT-PCR probes. PolII ChIP identified p1 and p2 (unannotated) as active promoters. (B) Increase in transcripts containing the indicated probe sequences relative to control (Mock) after RNAi treatment for five days. Mock control p1 transcripts are ~0.5% the level of RpL32 transcripts, p2 are ~0.6% and 3′ exon transcripts are ~1.7%. Error bars are standard errors, calculated using all RT-PCR replications from two independent experiments. Other times of RNAi treatment, other RNAi treatments (Nipped-B, Nipped-B combined with NELF and Spt5) and RNAi treatment times are shown in Figure S5. (C) ChIP using anti-Rpb3 and anti-Ser2P PolII antibodies five days after RNAi treatment. Error bars are standard errors calculated using all RT-PCR replicates from ChIP of two to three independent chromatin preparations. ChIP for Rad21 and Nipped-B binding after Rad21 and Spt5 depletion are shown in Figure S3.

Figure 6

Effects of Nipped-B, NELF-B and Spt5 Depletion on invected and engrailed Transcripts in BG3 Cells (A) Map of the invected-engrailed complex with ChIP profiles. The RT-PCR probes for both genes are in the 5′ exon (Table S1). (B) Changes in transcript levels five days after the indicated RNAi treatments. Mock control invected transcripts are ~0.1% the level of RpL32 transcripts, and engrailed transcripts are ~0.01%. Error bars are standard errors, calculated using all RT-PCR replications from two independent experiments. Additional days of treatment and Rad21 RNAi treatments are shown in Figure S7. Effects of Nipped-B, Rad21, NELF, and Spt5 depletion on E(spl)-C genes, which like invected and engrailed, are also PcG targets in BG3 cells [27], are shown in Figure S6. (C) PolII ChIP for invected after five days of Rad21 RNAi treatment. Error bars are standard errors calculated using all RT-PCR replicates from two to three independent chromatin preparations. The inv p1 probe covers nt 23-109 downstream from the TSS and inv exon A probe covers nt 691-802. ChIP for Rad21 and Nipped-B binding after Rad21 and Spt5 depletion are shown in Figure S3.

Figure 7

Model for Effects of Cohesin on Repressed Genes with Paused RNA Polymerase (A) Typical paused polymerase promoter with encircling cohesin, Nipped-B, upstream GAF, DSIF and NELF pausing factors, short nascent transcript (red), downstream GT repeats, and nucleosomes with H3K36me1. We theorize that GAF and the GU repeats in the initial nascent transcripts may independently recruit the pausing factors and Nipped-B, which then loads cohesin. (B) After phosphorylation of NELF, DSIF (Spt5), and PolII by activator-recruited pTEFb, NELF is released, unpausing PolII. At repressed genes, we posit that cohesin hinders transition of newly unpaused PolII to elongation, possibly by blocking association of Set2 methyltransferase with PolII, or inhibiting Set2 activity.