Histone Demethylase JMJD2B Coordinates H3K4/H3K9 Methylation and Promotes Hormonally Responsive Breast Carcinogenesis

Histone demethylase JMJD2B coordinates H3K4/H3K9 methylation and promotes hormonally responsive breast carcinogenesis Lei Shia,1, Luyang Suna,1, Qian Lia, Jing Lianga, Wenhua Yua, Xia Yia, Xiaohan Yanga, Yanyan Lia, Xiao Hana, Yu Zhanga, Chenghao Xuana, Zhi Yaob, and Yongfeng Shanga,b,2 aKey Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100191, China; and bTianjin Medical University, Tianjin 300070, China Edited* by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 23, 2011 (received for review November 23, 2010) It is well-documented that the methylation of histone H3 lysine and Trithorax) domain. In mammalian cells, multiple HMTs 4 (H3K4) and of H3K9 are mutually exclusive, an epigenetic have been characterized: SET1A and SET1B (the mammalian phenomenon conserved from yeast to humans. How this op- orthologs of yeast Set1), five mixed-lineage leukemia (MLL)- posed methylation modification is accomplished and coordinated family HMTs (MLL1–5),SMYD3,andSET7/9(14).Thein- in mammalian cells is poorly understood. Here we report that the creased complexity of the enzymatic machineries suggests that H3K9 trimethyl demethylase JMJD2B is an integral component of H3K4 methylation is biologically important and subject to the H3K4-specific methyltransferase, the mixed-lineage leukemia sophisticated controls in higher eukaryotes. Indeed, many (MLL) 2 complex. We show that theJMJD2B/MLL2complexis H3K4 methyltransferases in mammalian cells are essential copurified with estrogen receptor α (ERα) and is required for genes (15–17). ERα-regulated transcription. We demonstrate that H3K9 deme- The demethylation state of H3K9 is believed to be maintained thylation and H3K4 methylation are coordinated in ERα-activated by several members of the JmjC domain family of demethylases. transcription such that H3K9 demethylation is a prerequisite for Specifically, it has been reported that, in euchromatin, JHDM2A H3K4 methylation. Significantly, depletion of JMJD2B impairs the catalyzes the removal of mono- and dimethylation at H3K9 estrogen-induced G1/S transition of the cell cycle in vitro and (H3K9me1/2) (18) and JMJD2C acts to erase trimethylation at inhibits breast tumorigenesis in vivo. Interestingly, JMJD2B itself H3K9 (H3K9me3) (19), whereas JMJD2B has been shown to is an ERα target gene, and forms a feed-forward regulatory loop demethylate H3K9me3 at pericentric heterochromatin in mam- in regulation of the hormone response. Our results provide a mo- malian cells (20, 21). A role for JMJD2B in euchromatin func- lecular basis for the coordinated H3K4 methylation/H3K9 deme- tion has not yet been defined. thylation in transcription activation, link the trimethyl demethylase Here we report that the H3K9 trimethyl demethylase JMJD2B JMJD2B to euchromatin functions, and provide a mechanism for is physically associated with and an integral component of the JMJD2B in breast carcinogenesis. H3K4 methyltransferase MLL2 complex. We demonstrate that the JMJD2B/MLL2 complex interacts with ERα and is required histone methylation | breast cancer for the cellular function of ERα. We show that JMJD2B and the MLL2 complex interact to define the methylation status of H3K4 α ecent studies indicate that, analogous to other covalent his- and H3K9 in ER -activated transcription, in which H3K9 de- fi tone modifications such as acetylation, histone methylation is methylation precedes H3K4 methylation. In addition, we nd R α reversible. However, in contrast to histone acetylation, which is that JMJD2B itself is transcriptionally targeted by ER and may generally associated with active transcription, histone methyla- thus form a feed-forward regulatory loop in promoting hor- tion can be associated with either activation or repression of monally responsive breast carcinogenesis. MEDICAL SCIENCES transcription, depending on what effector protein is recruited Results and Discussion (1). Even within the same lysine residue, the biological con- sequences of methylation seem to be variable. For example, JMJD2B Is Physically Associated with the MLL2 Complex. To further methylation of histone H3 lysine 9 (H3K9), long considered understand the biological activity of JMJD2B and to investigate a hallmark of heterochromatin (2–8), was recently found to also its role, if any, in euchromatin functions, we generated a mam- mary carcinoma MCF-7 cell line that stably expresses FLAG- be present at the transcribed regions of active mammalian genes fi (9, 10), suggesting that certain methyl marks can have multiple tagged JMJD2B (FLAG-JMJD2B). Immunopuri cation of functions in the cell. JMJD2B from MCF-7 cell extracts with anti-FLAG and analysis Given the complexity of histone methylation modification, the of the JMJD2B-containing protein complex by mass spectrom- integration and/or coordination of methylation/demethylation in etry revealed that JMJD2B is associated with multiple com- a particular biological process becomes an issue of great im- ponents of the MLL2 complex (MLL2, ASH2L, RbBP5, and portance in further understanding the role of histone methylation in nucleosome functioning. Specifically, actively transcribed genes, including the ones that are regulated by estrogen receptor Author contributions: L. Shi, L. Sun, Q.L., J.L., W.Y., X. Yi, X. Yang, and Y.S. designed – research; L. Shi, L. Sun, Q.L., J.L., W.Y., X. Yi, X. Yang, Y.L., X.H., Y.Z., and C.X. performed (ER) (11 13), are marked by methylation at histone H3K4, but research; Z.Y. contributed new reagents/analytic tools; L. Shi, L. Sun, Q.L., J.L., W.Y., X. Yi, at the same time by demethylation at H3K9; H3K4 and H3K9 X. Yang, Y.L., X.H., Y.Z., C.X., Z.Y., and Y.S. analyzed data; and L. Shi, L. Sun, and Y.S. methylation levels are mutually exclusive, and this relationship is wrote the paper. conserved from fission yeast to humans. How opposed H3K4 The authors declare no conflict of interest. methylation and H3K9 demethylation are achieved and co- *This Direct Submission article had a prearranged editor. ordinated in transcription activation in mammalian cells is not 1L. Shi and L. Sun contributed equally to this work. fully understood. 2To whom correspondence should be addressed. E-mail: [email protected]. H3K4 methylation is deposited by a family of histone meth- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. yltransferases (HMT) that share a conserved SET (Su(var), E(z), 1073/pnas.1017374108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1017374108 PNAS | May 3, 2011 | vol. 108 | no. 18 | 7541–7546 Downloaded by guest on September 28, 2021 WDR5) as well as with several other proteins (hnRNP U, by coimmunoprecipitation experiments. Immunoprecipitation of HSP70, and ribosomal protein S9). Interestingly, four peptides MCF-7 cell extracts with antibodies against MLL2, WDR5, matching ERα were also identified in the JMJD2B-containing RbBP5, ASH2L, or JMJD2B followed by immunoblotting with protein complex (Fig. 1A and SI Appendix, Supplemental File 1). antibodies against ERα revealed that all components of the To confirm the in vivo association between JMJD2B and the JMJD2B/MLL2 complex including MLL2, but not MLL3, were MLL2 complex, coimmunoprecipitation experiments were per- coimmunoprecipitated with ERα (Fig. 2A Left). Reciprocally, formed with MCF-7 cell extracts. Immunoprecipitation with immunoprecipitation with antibodies against ERα followed by antibodies against JMJD2B followed by immunoblotting with immunoblotting with antibodies against MLL2, WDR5, RbBP5, antibodies against WDR5, RbBP5, ASH2L, or MLL2 demon- ASH2L, or JMJD2B demonstrated that ERα was associated with strated that JMJD2B was coimmunoprecipitated with all of these all of these proteins in vivo (Fig. 2A Right). components of the MLL2 complex, but not with JARID1B Next, we tested the effect of the JMJD2B/MLL2 complex on (PLU-1), another member of the JmjC domain-containing de- ERα-regulated transcription. To this end, MCF-7 cells were methylase, although JARID1B could be effectively immuno- transfected with specific siRNA molecules to knock down the precipitated with antibodies against HDAC1 (22) (Fig. 1B Left). expression of MLL2 or JMJD2B. The cells were deprived of Reciprocally, immunoprecipitation with antibodies against the estrogen for at least 3 d and treated with 17β-estradiol (E2). components of the MLL2 complex and immunoblotting with Real-time RT-PCR measurements revealed that knockdown of antibodies against JMJD2B also revealed that WDR5, RbBP5, either MLL2 or JMJD2B severely inhibited E2-stimulated ex- ASH2L, and MLL2, but not MLL1 and MLL4, were coimmu- pression of endogenous ERα target genes, including TFF1, noprecipitated with JMJD2B, although MLL1 and MLL4 could EBAG9, cathepsin D, and GREB1 (Fig. 2B), although to variable be efficiently coimmunoprecipitated with RbBP5, a subunit extents. The efficiency and specificity of MLL2 and JMJD2B shared by all of the MLL1–4 complexes (17, 23, 24) (Fig. 1B knockdown were examined by real-time quantitative RT-PCR Right). A weak interaction between JMJD2B and MLL3 was also and/or Western blotting analysis (Fig. 2C). These results indicate detected (Fig. 1B Lower Right). Collectively, these results sup- that the JMJD2B/MLL2 complex is required for ERα-activated port the suggestion that JMJD2B is physically associated with the gene transcription. MLL2 complex in vivo. To further support this

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