Global identification of MLL2-targeted loci reveals MLL2’s role in diverse signaling pathways Changcun Guoa,b,c,1, Chun-Chi Changa,b,c,1, Matthew Worthama,b,c, Lee H. Chena,b,c, Dawn N. Kernagisc, Xiaoxia Qind, Young-Wook Choe,2, Jen-Tsan Chid, Gerald A. Granta,b,f, Roger E. McLendona,b,c, Hai Yana,b,c, Kai Gee, Nickolas Papadopoulosg, Darell D. Bignera,b,c, and Yiping Hea,b,c,3 aThe Preston Robert Tisch Brain Tumor Center at Duke, bPediatric Brain Tumor Foundation Institute, cDepartment of Pathology, dInstitute for Genome Sciences and Policy, and fDepartment of Surgery, Duke University, Durham, NC 27710; eLaboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and gLudwig Center for Cancer Genetics and Therapeutics, Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21231 Edited* by Bert Vogelstein, Johns Hopkins University, Baltimore, MD, and approved September 14, 2012 (received for review June 1, 2012) Myeloid/lymphoid or mixed-lineage leukemia (MLL)-family genes SWI/SNF, the MLL2 or MLL3 (hereafter referred to as MLL2/ encode histone lysine methyltransferases that play important 3) complex has been found to play essential roles as a coactivator roles in epigenetic regulation of gene transcription. MLL genes for transcriptional activation by nuclear hormone receptors (11). are frequently mutated in human cancers. Unlike MLL1, MLL2 (also Consistent with this notion, previous studies have shown that known as ALR/MLL4) and its homolog MLL3 are not well-under- MLL2/3 complexes regulate Hox gene transcription in an es- stood. Specifically, little is known regarding the extent of global trogen receptor-dependent manner, and that they play critical MLL2 involvement in the regulation of gene expression and the roles in PPARγ-dependent adipogenesis (12–14). mechanism underlying its alterations in driving tumorigenesis. Somatically acquired epigenetic changes are prevalent in hu- Here we profile the global loci targeted by MLL2. A combinatorial man cancers. Recent cancer genetics studies have uncovered analysis of the MLL2 binding profile and gene expression in MLL2 frequent somatic loss-of-function mutations in the genes encod- wild-type versus MLL2-null isogenic cell lines identified direct tran- ing MLL2/3 complex subunits in a variety of cancer types. UTX scriptional target genes and revealed the connection of MLL2 to was found to be mutated in numerous human cancer types (15– multiple cellular signaling pathways, including the p53 pathway, 17). MLL3 was found to be mutated in a subset of colorectal cAMP-mediated signaling, and cholestasis signaling. In particular, cancers and in transitional cell carcinoma (16, 18–20). Most nota- we demonstrate that MLL2 participates in retinoic acid receptor bly, several recent studies identified frequent MLL2-inactivating signaling by promoting retinoic acid-responsive gene transcrip- mutations in non-Hodgkin B-cell lymphomas (21–23). Finally, tion. Our results present a genome-wide integrative analysis of genes encoding MLL2/3 complex subunits, particularly MLL2, the MLL2 target loci and suggest potential mechanisms underlying MLL3,andUTX, were found to be mutated in medulloblastoma, tumorigenesis driven by MLL2 alterations. establishing them as critical tumor suppressors (24–26). In contrast to the compelling genetic evidence, much less is tumor suppressor | somatic targeting | S100A gene cluster known regarding the mechanism underlying the tumor suppres- sor roles of the MLL2/3 complex. It was shown that knockdown odulation of chromatin accessibility through chromatin of MLL2 resulted in reduced cancer cell growth and altered Mremodeling and histone modification is a critical step in adhesion in HeLa cells (9). An MLL3-knockout mouse model regulating eukaryotic gene transcription. Histone H3 lysine 4 study showed that MLL3 plays a role in DNA damage response (H3K4) methylation by histone methyltransferases is an evolu- in a p53-dependent manner, suggesting that the complex serves tionarily conserved epigenetic mark for active gene transcription as a transcriptional coactivator for p53 (27). The link to p53 is (1). In mammalian cells, six complexes with H3K4 methyl- consistent with the tumor suppressor role of the MLL3 complex. transferase activity have been identified, each with one SET However, it is most likely that MLL2/3 epigenetic complexes domain-containing methyltransferase subunit, including Set1A, have a broader role biochemically and that the deficiency of the Set1B, and four MLL-family proteins. In humans, these MLL- pathway has a more profound impact on cellular processes. To family genes include MLL1, MLL2 (GenBank accession no. further elucidate the tumor suppressor role of MLL2, we per- NM_003482; also known as ALR/MLL4), MLL3, and MLL4 formed a direct profiling of the global loci that are targeted by (GenBank accession no. NM_014727; also known as MLL2 or MLL2. Moreover, we show that somatic knockout of MLL2 in Wbp7, conflicting with the official symbol for MLL2). Each of human cells affects the expression of a variety of genes. We these proteins is associated with a complex that modulates his- note a set of genes that are retinoic acid-responsive and may be tone methylation (2). Among the MLL genes, MLL1 and its directly regulated by MLL2. Finally, integrative pathway anal- homolog MLL4 have been the most extensively studied, as the ysis uncovered various signaling pathways that are regulated by former is frequently involved in genetic alterations in leukemia the MLL2 complex. Our results reveal the global and pathway- (reviewed in ref. 3). In addition, MLL4 has been found to be overexpressed in breast and colorectal cell lines (4). MLL1 and MLL4 play essential roles in regulating the expression of genes Author contributions: C.G. and Y.H. designed research; C.G., C.-C.C., and Y.H. performed research; C.-C.C., Y.-W.C., G.A.G., K.G., N.P., and D.D.B. contributed new reagents/analytic that are involved in cell differentiation and early embryonic tools; C.G., C.-C.C., M.W., L.H.C., D.N.K., X.Q., J.-T.C., R.E.M., H.Y., N.P., and Y.H. analyzed development, among which the most notable and best-estab- data; and C.G. and Y.H. wrote the paper. MEDICAL SCIENCES lished are the Homeobox (Hox) genes (5, 6). The authors declare no conflict of interest. MLL2 was originally cloned as a human homolog of Dro- *This Direct Submission article had a prearranged editor. sophila trithorax (7). MLL2 and its homolog, MLL3, function 1C.G. and C.-C.C. contributed equally to this work. distinctly from MLL1 and MLL4. Each of them associates with 2Present address: Korea Basic Science Institute Chuncheon Center, Chuncheon-si 200-701, nuclear receptor coactivator 6 (also known as activating signaling Republic of Korea. cointegrator 2) to form a complex that contains other subunits 3To whom correspondence should be addressed. E-mail: [email protected]. including ASH2L, RbBP5, WDR5, DPY30, PTIP, PA1, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. UTX (8–10). Together with the chromatin-remodeling complex 1073/pnas.1208807109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1208807109 PNAS | October 23, 2012 | vol. 109 | no. 43 | 17603–17608 Downloaded by guest on September 27, 2021 specific roles of MLL2 and suggest that it participates in reg- (30), we have established a human cell assay that consists of ulating a wide range of pathways with relevance to its role in isogenic cell lines that differ only in the presence or absence of oncogenesis. an MLL2-Flag allele, thus permitting the direct profiling of MLL2- targeted loci by a proven-quality antibody-based ChIP-seq. Results Generation of Endogenous Flag-Tagged MLL2-Expressing Cells. Global Identification of MLL2-Targeted Loci. The parental cell line Identifying genomic binding sites is a powerful strategy to re- and MLL2-Flag–expressing cells (clone MLL2-Flag1 and 2) were veal genes regulated by transcriptional regulators (5, 28, 29). subjected to anti-Flag antibody-based ChIP-seq procedures (Fig. However, two hurdles confront the direct global profiling of S2A). We modified a previously described differential tag-density MLL2-targeted loci: (i) It is a massive protein of 5,537 amino analysis (34, 35) to obtain a global view of MLL2-bound loci. We acids (∼600 kDa), which complicates biochemical studies that generated bins (regions) across the human genome and com- require ectopic expression; and (ii) neither a proven-quality pared the tag density for each bin between the parental and antibody nor a human cell assay is available for chromatin im- MLL2-Flag–expressing cell lines. The relative tag density of the munoprecipitation-sequencing (ChIP-seq). To overcome these MLL2-Flag–expressing cell line to the parental line showed fre- obstacles, we have adopted a somatic knockin-based approach in quent spikes (Fig. 1C), indicating an MLL2-Flag–specific enrich- which an endogenous protein is fused with a short tagging pep- ment. In contrast, such enrichment was absent in the parental tide, which facilitates antibody-based ChIP studies (30, 31). We line, as indicated by the reverse tag-density ratio (Fig. 1C). The chose the HCT116 carcinoma cell line for our study, as it is same set of MLL2-Flag–specific enrichments was also apparent a near-diploid cell line with well-defined key cancerous pathways. in MLL2-Flag–expressing cell line 2 (Fig. S2B). Furthermore, this cell line has homozygous MLL3-inactivating To identify MLL2-targeted regions, we applied a criterion by mutations, thus preventing compensatory effects from obscuring which at least fivefold enrichment was required to identify dif- functional analysis (19). An rAAV-based somatic knockin con- ferential tag density (35). A total of 2,060 MLL2 binding events struct (30, 32, 33) enabled the insertion of a DNA fragment fi encoding Flag tags immediately upstream of the stop codon of were identi ed.