Short Rnas Are Transcribed from Repressed Polycomb Target Genes and Interact with Polycomb Repressive Complex-2
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Short RNAs Are Transcribed from Repressed Polycomb Target Genes and Interact with Polycomb Repressive Complex-2 The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Kanhere, Aditi, Keijo Viiri, Carla C. Araújo, Jane Rasaiyaah, Russell D. Bouwman, Warren A. Whyte, C. Filipe Pereira, et al. “Short RNAs Are Transcribed from Repressed Polycomb Target Genes and Interact with Polycomb Repressive Complex-2.” Molecular Cell 38, no. 5 (June 2010): 675–688. © 2010 Elsevier Inc. As Published http://dx.doi.org/10.1016/j.molcel.2010.03.019 Publisher Elsevier Version Final published version Citable link http://hdl.handle.net/1721.1/96323 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Molecular Cell Article Short RNAs Are Transcribed from Repressed Polycomb Target Genes and Interact with Polycomb Repressive Complex-2 Aditi Kanhere,1 Keijo Viiri,1 Carla C. Arau´ jo,1 Jane Rasaiyaah,1 Russell D. Bouwman,1 Warren A. Whyte,2,3 C. Filipe Pereira,4 Emily Brookes,4 Kimberly Walker,2 George W. Bell,2 Ana Pombo,4 Amanda G. Fisher,4 Richard A. Young,2,3 and Richard G. Jenner1,* 1Division of Infection and Immunity, University College London, London W1T 4JF, UK 2Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02141, USA 4MRC Clinical Sciences Centre, Imperial College London, London W12 0NN, UK *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.03.019 SUMMARY of hundreds of developmental regulators in ES cells that would otherwise induce cell differentiation (Azuara et al., 2006; Boyer Polycomb proteins maintain cell identity by repres- et al., 2006; Lee et al., 2006). In differentiated cells, activated sing the expression of developmental regulators genes important for the identity of those cells lose H3K27me3, specific for other cell types. Polycomb repressive whereas genes that regulate alternate cellular identities remain complex-2 (PRC2) catalyzes trimethylation of histone methylated and repressed (Bernstein et al., 2006a; Lee et al., H3 lysine-27 (H3K27me3). Although repressed, PRC2 2006; Mikkelsen et al., 2007; Roh et al., 2006). targets are generally associated with the transcrip- Although repressed, PRC2 target genes are thought to adopt a poised state that allows their rapid upregulation upon ES cell tional initiation marker H3K4me3, but the signifi- differentiation (Boyer et al., 2006; Lee et al., 2006). This poised cance of this remains unclear. Here, we identify a state is reflected by the association of PRC2 target genes with class of short RNAs, 50–200 nucleotides in length, histone H3K4me3, a marker of transcriptional initiation (Azuara 0 transcribed from the 5 end of polycomb target genes et al., 2006; Bernstein et al., 2006a; Roh et al., 2006). The in primary T cells and embryonic stem cells. Short unphosphorylated and Ser-5 phosphorylated forms of RNA RNA transcription is associated with RNA poly- polymerase (pol) II have also been detected at some polycomb merase II and H3K4me3, occurs in the absence of target genes, but not the Ser-2 phosphorylated form that is asso- mRNA transcription, and is independent of poly- ciated with transcriptional elongation (Dellino et al., 2004; Lee comb activity. Short RNAs form stem-loop structures et al., 2006; Stock et al., 2007). The block to RNA pol II elongation resembling PRC2 binding sites in Xist, interact with at these genes is due, at least in part, to the activity of PRC1, PRC2 through SUZ12, cause gene repression in cis, which binds to H3K27me3. PRC1 contains Ring1 that ubiquiti- nates H2A, blocking elongation by RNA pol II (Stock et al., and are depleted from polycomb target genes acti- 2007; Zhou et al., 2008). vated during cell differentiation. We propose that PRC2 is often associated with CpG islands (Lee et al., 2006; short RNAs play a role in the association of PRC2 Ku et al., 2008), but PRC2 has no known DNA sequence binding with its target genes. specificity and it is not clear how PRC2 associates with its target genes in mammals. Recent results have shown that PRC2 can interact with long non-coding (nc)RNA transcripts. PRC2 inter- INTRODUCTION acts with the 1.6 kb ncRNA RepA transcribed from Xist and this is necessary for H3K27me3 and X chromosome inactivation Polycomb group (PcG) proteins are essential for embryogenesis (Zhao et al., 2008). Similarly, the ncRNA HOTAIR associates with and for maintaining embryonic stem (ES) cell pluripotency and PRC2 and induces methylation of the HOXD locus in trans (Rinn differentiated cell states (Boyer et al., 2006; Faust et al., 1995; et al., 2007) and the antisense transcript Kcnq1ot1 associates Lee et al., 2006; O’Carroll et al., 2001; Pasini et al., 2007; van with PRC2 and induces methylation of Kcnq1 (Pandey et al., der Stoop et al., 2008). PcG proteins form at least two polycomb 2008). repressive complexes (PRCs) that are conserved across Meta- Because polycomb target genes show evidence of stalled zoa. PRC2 catalyzes trimethylation of lysine 27 of histone H3 RNA polymerase II and do not transcribe appreciable amounts (H3K27me3), forming a binding site for PRC1 (Cao et al., 2002; of mRNA, we hypothesized that short RNAs may be tran- Wang et al., 2004). scribed from these genes. We describe here the identification Genome-wide measurements of PRC2 localization and H3K27 of such a set of short RNAs transcribed from the 50 ends of methylation have revealed that PRC2 represses the expression polycomb target genes in primary human CD4+ T cells and in Molecular Cell 38, 675–688, June 11, 2010 ª2010 Elsevier Inc. 675 Molecular Cell Polycomb-Associated Short RNAs A Figure 1. Detection of Promoter-Associated Short RNAs in Primary Human T Cells (A) Experimental strategy. Short RNA was purified from human CD4+ T cells, polyadenylated, ampli- fied, and labeled with Cy5 and hybridized together Gene x Short RNA with Cy3-labeled mRNA (experiment 1) or total long RNA (experiment 2) to a custom microarray con- taining probes for the promoters, 50 exons, first intron, and 30 exons of human RefSeq genes, CD4+ T-cells Microarray Probes for together with control probes for snRNA and each gene snoRNAs. (B) Background-subtracted array signals for Long RNA control snRNA and snoRNA probes in the mRNA channel (x axis) and the short RNA channel (y axis). B C (C) Fraction of promoter regions (1 kb upstream of 5 30 10 Promoter mRNA transcription start site), 50 exons (up to 1kb 5' Exon downstream of mRNA 50 end), and first introns 25 l 104 1st Intron that give rise to short RNAs. a n (D) Distribution of short RNAs relative to the TSS of ig ) 20 s 3 % the average gene, plotted as a moving average with ( A A 10 s N a window of 10 bp. e 15 R n t e (E) Venn diagram showing the overlap between r 102 o G 10 genes with associated short RNAs detected in Sh 10 this study compared with two previous studies 5 (Kapranov et al., 2007; Seila et al., 2008). 0 2 3 4 5 10 10 10 10 10 Replicate 1 Replicate 2 Both mRNA signal D 1000 E Seila et al. (2008) known short nuclear (sn)RNA and short nucleolar (sno)RNA that acted as positive l a n 800 controls (Figure 1B and Figure S1). g i 517 s We investigated whether we could A 2973 N 600 detect short RNAs transcribed from R t r protein-coding genes. Applying a thresh- o 102 h 400 758 old derived from our snRNA probes, we S 7987 e found thousands of short RNAs tran- g a r 200 573 scribed from the sense strands of pro- e v 1752 A moters, exons, and introns of protein- 0 coding genes in replicate experiments -2500 0 2500 5000 This study (Figure 1C, Table S2). The RNAs were 0 Average gene Kapranov et al. concentrated at the 5 end of genes, within (2007) 700 bp upstream and downstream of the mRNA TSS (Figure 1D). A substantial subset of the genes associated with short RNAs in primary human CD4+ T cells also murine ES cells and present evidence for their interaction with produced short RNAs in cell lines and ES cells (Kapranov et al., PRC2. 2007; Seila et al., 2008)(Figure 1E), although often from different locations (Figure S1). These results indicate that short RNAs RESULTS are generated in primary human T cells, are most abundant in promoter-proximal sequences, and are a general feature of the Detection of Short RNAs Associated with the 50 End transcriptome of normal somatic cells. of Genes We designed a DNA microarray to identify short RNAs at the 50 Transcription of Short RNAs from Genes end of human genes. RNA from primary human CD4+ T cells that Are Otherwise Repressed was fractionated by size into short (<200 nt) and large RNAs, The short RNAs described in previous reports are associated labeled with fluorescent dyes, and hybridized to the microarrays with transcriptionally active genes but not with repressed poly- in replicate experiments (Figure 1A, and see Figure S1 available comb target genes (Affymetrix/CSHL ENCODE Transcriptome online). The arrays contained multiple probes allowing detection Project, 2009; Core et al., 2008; Kapranov et al., 2007; Seila of sense-strand RNA at the promoter, 50-most exons, first intron et al., 2008; Taft et al., 2009). We therefore examined the tran- and 30-most exons of protein-coding genes (Supplemental Infor- scriptional status of T cell genes that produce short RNAs.