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Setdb1 Contributes to Repression of Genes Encoding Developmental Regulators and Maintenance of ES Cell State

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Setdb1 Contributes to Repression of Genes Encoding Developmental Regulators and Maintenance of ES Cell State Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION Cole et al. 2008; Dejosez et al. 2008; Jiang et al. 2008; Kim SetDB1 contributes to et al. 2008; Marson et al. 2008; Tam et al. 2008). These repression of genes encoding key transcriptional regulators occupy the promoters of actively transcribed genes encoding transcription factors, developmental regulators and signaling components, and chromatin-modifying en- maintenance of ES cell state zymes that promote ES cell self-renewal. They also oc- cupy genes encoding a large set of developmental regula- Steve Bilodeau,1,3 Michael H. Kagey,1,3 tors that are silent in ES cells, but whose expression is Garrett M. Frampton,1,2 Peter B. Rahl,1 and associated with lineage commitment and cellular dif- 1,2,4 ferentiation. Polycomb-repressive complexes (PRCs) co- Richard A. Young occupy the genes encoding these developmental regula- 1Whitehead Institute for Biomedical Research, Cambridge, tors to help maintain a silent transcriptional state in ES Massachusetts 02142, USA; 2Department of Biology, cells (Azuara et al. 2006; Boyer et al. 2006; Lee et al. 2006; Massachusetts Institute of Technology (MIT), Stock et al. 2007; van der Stoop et al. 2008). Cambridge, Massachusetts 02139, USA The regulation of chromatin structure, which includes nucleosome remodeling and post-translational modifica- Transcription factors that play key roles in regulating tion of histone proteins, is necessary for the establish- embryonic stem (ES) cell state have been identified, but ment and maintenance of heritable gene expression the chromatin regulators that help maintain ES cells are patterns during development (Workman 2006; Dunn and less well understood. A high-throughput shRNA screen Kingston 2007; Kouzarides 2007; Surani et al. 2007). Trithorax group (TrxG) and Polycomb group (PcG) protein was used to identify novel chromatin regulators that complexes are key regulators of chromatin structure that influence ES cell state. Loss of histone H3 Lys 9 (H3K9) are necessary for maintenance of the ES cell gene expres- methyltransferases, particularly SetDB1, had the most sion program and are required for segmental identity in profound effects on ES cells. Chromatin immunoprecip- the developing embryo (for review, see Schuettengruber itation (ChIP) coupled with massively parallel DNA se- et al. 2007). TrxG complexes catalyze histone H3 Lys 4 quencing (ChIP-Seq) and functional analysis revealed trimethylation (H3K4me3) in proximity to promoters of that SetDB1 and histone H3K9-methylated nucleosomes genes that experience transcription initiation by RNA occupy and repress genes encoding developmental regu- polymerase II. Since most genes experience some level lators. These SetDB1-occupied genes are a subset of the of transcription initiation, most promoters are occupied ‘‘bivalent’’ genes, which contain nucleosomes with by nucleosomes with histone H3K4me3 (Guenther H3K4me3 (H3K4 trimethylation) and H3K27me3 modi- et al. 2007). The PcG protein complex PRC2 catalyzes fications catalyzed by Trithorax and Polycomb group H3K27me3, which contributes to repression of genes en- proteins, respectively. These genes are subjected to re- coding key developmental regulators. Disruption of PcG pression by both Polycomb group proteins and SetDB1, function in ES cells leads to derepression of these develop- and loss of either regulator can destabilize ES cell state. mental genes, alteration of the ES cell transcriptional pro- gram, and a loss of the ES cell state (Boyer et al. 2006; Lee Supplemental material is available at http://www.genesdev.org. et al. 2006; Pasini et al. 2007; Stock et al. 2007; Jaenisch and Young 2008; van der Stoop et al. 2008). In ES cells, Received June 26, 2009; revised version accepted September genes encoding lineage-specific developmental regula- 10, 2009. tors contain nucleosomes with both H3K4me3 and H3K27me3 modifications, and have thus been called ‘‘bivalent’’ (Bernstein et al. 2006). These genes apparently Embryonic stem (ES) cells provide an important model experience some level of transcription initiation, and system to study the control of early development and thus TrxG-mediated histone H3K4 methylation, but hold significant potential for clinical therapies because are silenced by PcG complexes, which may occur through of their distinctive capacity to both self-renew and dif- repression of transcription elongation (Stock et al. 2007). ferentiate into multiple lineages (Thomson et al. 1998; Although many chromatin remodeling and modifying Reubinoff et al. 2000; Pera and Trounson 2004; Keller activities have been described, only a few are known 2005; Hochedlinger and Jaenisch 2006; Yang and Smith to contribute to the control of ES cell state. We used 2007). Studies of key ES cell transcription factors— a screening approach with a shRNA library to screen including Oct4, Sox2, Nanog, Ronin, Tcf3, and others— for additional chromatin regulators of ES cell state. have led to models for ES cell transcriptional regula- Loss of histone H3K9 methyltransferases as well as tory circuitry that can account for several important PcG components was found to cause loss of ES cell features of the gene expression program of these unique state in this screen. Further study of the H3K9 methyl- cells (Boyer et al. 2005; Loh et al. 2006; Chen et al. 2008; transferase SetDB1 revealed that it contributes to re- pression of a subset of genes encoding developmental [Keywords: SetDB1; ES cell; H3K9 methylation; polycomb; repression; regulators in ES cells. These results indicate that there is pluripotency] an additional layer of chromatin-mediated repression at 3 These authors contributed equally to this work. these developmental genes, and that H3K9me3-modified 4Corresponding author. E-MAIL [email protected]; FAX (617) 258-9872. nucleosomes are not exclusive to constitutive hetero- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1837309. chromatin. 2484 GENES & DEVELOPMENT 23:2484–2489 Ó 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press SetDB1 represses developmental regulators Results and Discussion cells in each well was measured. Control shRNAs target- ing Oct4 and Stat3, positive regulators of pluripotency in In order to identify chromatin regulators that may be mES cells (Nichols et al. 1998; Niwa et al. 1998; Hay et al. involved in the establishment or maintenance of cell 2004), produced cells that lost Oct4 signal, as expected state, we screened murine ES (mES) cells with a shRNA (Fig. 1B). In contrast, a control shRNA designed against library containing four to six hairpins directed against Tcf3, a negative regulator of Oct4 and Nanog (Pereira each of 197 chromatin regulators (Fig. 1A). ES cells were et al. 2006; Cole et al. 2008; Tam et al. 2008), caused an seeded into 384-well plates, and each well was infected increase in Oct4 staining. To normalize for plate variabil- with an individual lentiviral construct delivering ity, the average Oct4 staining intensity for the cells was a shRNA targeting a chromatin regulator. After selecting used to calculate a Z-score for each well, taking into for stable integration of the shRNA construct, the cells account values for negative control infections on each were fixed, and the average Oct4 staining intensity of the plate (see the Supplemental Material). The majority of the shRNAs targeting chromatin regulators produced Z-scores that were distributed between those observed for shRNAs targeting Oct4 and Tcf3, as expected (Fig. 1C; Supplemental Tables S1, S2). The chromatin regulators whose knockdowns reduced Oct4 levels nearly as profoundly as knockdown of Oct4 itself were of particular interest because many have been implicated in gene silencing, and many of these are associated with histone H3K9 and H3K27 methylation (Fig. 1C; Supplemental Table S1). The chromatin regula- tors associated with H3K9 methylation include SetDB1/ ESET, Ube2i/Ubc9, Ehmt1, and Suv39h2. Those associ- ated with H3K27 methylation include the Polycomb components Cbx7, Cbx8/Pc3, and Ezh2, and their iden- tification in this screen is consistent with previous evidence implicating Polycomb in control of ES cell state (Schuettengruber et al. 2007). The loss of the histone deacetylase Hdac3 and the Sin3/HDAC complex compo- nents Sin3a and Sap18 had similar effects. The loss of certain chromatin regulators led to increased Oct4 levels, and these included three members of the SWI/SNF family (Smarcd1, Arid1a, and Smarcb1) (Fig. 1C). Thus, the re- sults of this screen implicate multiple chromatin regula- tors in control of ES cell state. One of the largest classes of chromatin regulators identified in the screen was associated with histone H3K9 methylation. From this class we selected the SetDB1 H3K9 methyltransferase (Schultz et al. 2002; Ayyanathan et al. 2003; Wang et al. 2003) for further detailed study because its loss produced the most pro- found effects on ES cell state (Fig. 1C; Supplemental Table S1; Supplemental Fig. S1) and SetDB1-null blastocysts have deficiencies in the inner cell mass (Dodge et al. Figure 1. Identification of chromatin regulators of ES cell state. (A) 2004). Independent experiments demonstrated that mul- Outline of the screening protocol. mES cells were seeded without a mouse embryonic fibroblast (MEF) feeder layer into 384-well tiple shRNAs
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