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Methylation of RUNX1 by PRMT1 Abrogates SIN3A Binding and Potentiates Its Transcriptional Activity

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Methylation of RUNX1 by PRMT1 Abrogates SIN3A Binding and Potentiates Its Transcriptional Activity Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity Xinyang Zhao,1 Vladimir Jankovic,1 Alexander Gural,1 Gang Huang,1 Animesh Pardanani,1 Silvia Menendez,1 Jin Zhang,1 Richard Dunne,1 Andrew Xiao,2 Hediye Erdjument-Bromage,1 C. David Allis,2 Paul Tempst,1 and Stephen D. Nimer1,3 1Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA; 2Rockefeller University, New York, New York 10021, USA RUNX1/AML1 is required for the development of definitive hematopoiesis, and its activity is altered by mutations, deletions, and chromosome translocations in human acute leukemia. RUNX1 function can be regulated by post-translational modifications and protein–protein interactions. We show that RUNX1 is arginine-methylated in vivo by the arginine methyltransferase PRMT1, and that PRMT1 serves as a transcriptional coactivator for RUNX1 function. Using mass spectrometry, and a methyl-arginine-specific antibody, we identified two arginine residues (R206 and R210) within the region of RUNX1 that interact with the corepressor SIN3A and are methylated by PRMT1. PRMT1- dependent methylation of RUNX1 at these arginine residues abrogates its association with SIN3A, whereas shRNA against PRMT1 (or use of a methyltransferase inhibitor) enhances this association. We find arginine-methylated RUNX1 on the promoters of two bona fide RUNX1 target genes, CD41 and PU.1 and show that shRNA against PRMT1 or RUNX1 down-regulates their expression. These arginine methylation sites and the dynamic regulation of corepressor binding are lost in the leukemia-associated RUNX1–ETO fusion protein, which likely contributes to its dominant inhibitory activity. [Keywords: CD41; PU.1; arginine methylation; myeloid differentiation; AML1 target genes] Supplemental material is available at http://www.genesdev.org. Received November 8, 2007; revised version accepted December 28, 2007. The RUNX1/CBF␤ transcriptional regulatory complex is RUNX1c (which contains 480 amino acids). All contain required for the development of murine definitive hema- the Runt DNA-binding domain, and both RUNX1b and topoiesis (Okuda et al. 1996; Wang et al. 1996). RUNX1 RUNX1c contain transcriptional activating domains. (also known as AML1, CBFA2, and PEBP2␣B) is the There is no apparent functional difference between DNA-binding component of this complex, and its func- RUNX1b and RUNX1c despite their alternative N ter- tion is often compromised by mutations or chromosom- mini. (See Supplemental Fig. S1 for a sequence align- al translocations in acute leukemia. The RUNX1–ETO ment. In this paper, we used amino acid number accord- fusion transcription factor, which is generated by the ing to RUNX1b.) RUNX1 can act as either a transcrip- t(8;21) in acute myeloid leukemia (AML), has aberrant tional activator or a repressor, depending on the cellular transactivating properties and can function as a domi- and promoter context. RUNX1 often activates transcrip- nant inhibitor of RUNX1 function and of other transcrip- tion weakly, but its interactions with other transcription tion factors as well (Mao et al. 1999; Nimer and Moore factors such as GATA-1, ETS-1, and C/EBP␣ enhance its 2004; Zhang et al. 2004). RUNX1–ETO can promote the activating properties (Wotton et al. 1994; Zhang et al. self-renewal of human and murine hematopoietic stem 1996; Elagib et al. 2003). To achieve transcriptional ac- cells, which may increase the chance of developing sec- tivation, RUNX1 recruits coactivator molecules, such as ondary mutations, leading ultimately to the develop- ALY, p300, YAP, and MOZ (Bruhn et al. 1997; Kitabaya- ment of acute leukemia (Higuchi et al. 2002; Mulloy et shi et al. 1998, 2001; Yagi et al. 1999), which generally al. 2003). bind to the C terminus of RUNX1. However, RUNX1 The human RUNX1 gene has three different isoforms, can specifically silence CD4 gene expression during ma- RUNX1a (which encodes a theoretical 250-amino-acid ture T-cell differentiation (Taniuchi et al. 2002); its re- protein), RUNX1b (which contains 453 amino acids), and pression of the p21 gene promoter in NIH3T3 cells has also been reported (Lutterbach et al. 2000). Although two 3Corresponding author. E-MAIL [email protected]; FAX (212) 794-5849. distinct repressor complexes bind to RUNX1, the core- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1632608. pressor SIN3A complex (Lutterbach et al. 2000), and the 640 GENES & DEVELOPMENT 22:640–653 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Arginine methylation of Runx1 Groucho/TLE repressor complex (Levanon et al. 1998), RUNX1 on both the IL-3 promoter (data not shown) and the precise mechanism through which specific genes are the CD41 promoter (Supplemental Fig. S2). Both MTA (a silenced by RUNX1 is not well understood. nonspecific methyltransferase inhibitor) and shRNA RUNX1 is modified by phosphorylation (e.g., by the that knock down PRMT1 levels (Rezai-Zadeh et al. 2003) serine/threonine kinase ERK2) (Tanaka et al. 1996) and abrogate promoter activation by RUNX1. by acetylation (e.g., by the coactivator acetyltransferase As these findings could reflect global effects of PRMT1 p300) (Yamaguchi et al. 2004). Both modifications en- on transcription, we first examined whether PRMT1 hance transcriptional activation by RUNX1, thereby in- physically interacts with RUNX1 in vivo. Using HEL fluencing its role in hematopoiesis. Recently, interac- cell extracts, we readily coimmunoprecipitated PRMT1 tions of RUNX1 with the lysine methyltransferase using an anti-RUNX1 antibody and immunoprecipitated SUV39H1 have been reported (Chakraborty et al. 2003; RUNX1 protein using an anti-PRMT1 antibody, under Reed-Inderbitzin et al. 2006), and a role for this methyl- stringent washing conditions (900 mM NaCl and 1% transferase in gene repression by RUNX1 has been sug- NP-40) (Fig. 1A,B). As PRMT1 is involved in RNA export gested. (Yu et al. 2004) and can bind DNA via sequence-specific The arginine methylation of nonhistone proteins is be- transcription factors (Rezai-Zadeh et al. 2003), we in- ing increasingly identified. PRMT1 is an arginine meth- cluded RNase A and ethidium bromide in these immu- yltransferase that functions to monomethylate or asym- noprecipitation reactions to exclude the possibility that metrically dimethylate arginine residues. PRMT1 is its interactions with RUNX1 are mediated through RNA found in both the cytoplasm and the nucleus (Herrmann or DNA (Lai and Herr 1992). Neither treatment dis- et al. 2005), and it accounts for most of the Type I argi- rupted the RUNX1–PRMT1 immunoprecipitable com- nine methyltransferase activity in cells (Tang et al. plex, showing that PRMT1 and RUNX1 interact in a 2000). It is well established that arginine methylation by DNA- and RNA-independent manner. PRMT1 serves as a general marker for active transcrip- To map the region in RUNX1 that interacts with tion (Huang et al. 2005). PRMT1 targets histone H4R3 PRMT1, we used various GST-RUNX1 fusion proteins for arginine methylation, promoting the subsequent (∼5 µg of protein per pull-down reaction) to pull down in p300- and CARM1-mediated acetylation and methyla- vitro translated PRMT1 (Supplemental Fig. S3). Clearly, tion of histone tails (An et al. 2004). It also modifies several different portions of the C terminus of RUNX1 coactivator molecules such as PGC-1 (Teyssier et al. can pull down PRMT1, which is similar to fibrillarin, a 2005) and NIP45 (Mowen et al. 2004), and it is recruited protein that interacts with PRMT1 through several re- by YY1 (Rezai-Zadeh et al. 2003), by nuclear hormone gions (Yanagida et al. 2004). However, neither the Runt receptors (Wang et al. 2001), and by p53 for transcrip- domain itself (GST-RUNX1 60–182) nor the N-terminal tional activation (An et al. 2004). region of RUNX1 (GST-RUNX1 2–60) pulls down RUNX proteins contain several potential arginine PRMT1. Thus, the binding of PRMT1 can occur via more methylation sites, which prompted us to examine than one domain within the RUNX1 C-terminal region. whether PRMT1 can methylate RUNX1 and alter its ef- To further demonstrate the interaction of these proteins fect on gene expression. We show that PRMT1 directly in vitro, we also used GST-PRMT1, which readily pulled interacts with RUNX1, methylates RUNX1, and func- down in vitro translated RUNX1 (Supplemental Fig. S3C). tions as a coactivator for RUNX1-dependent transcrip- tional activation; and that reduction in the level of Multiple sites in RUNX1 are arginine-methylated in PRMT1 (or RUNX1) lowers the expression of several vitro by PRMT1 true RUNX1 target genes. While several sites within RUNX1 appear to be methylated, methylation of argi- PRMT1 does target nonhistone proteins for arginine nine residues within an RTAMR region of the RUNX1, methylation (Boisvert et al. 2003), so we determined if just C-terminal to the Runt DNA-binding domain, RUNX1 is arginine-methylated by PRMT1 in vitro and abrogates the binding of SIN3A, thereby promoting mapped the sites of methylation using mass spectrom- RUNX1 transcriptional activity. The RTAMR region is etry. By incubating in vitro translated RUNX1 with re- not present in RUNX1–ETO, which binds SIN3A in a combinant GST-PRMT1 and 3H-methyl-SAM, we methylation-insensitive manner. We find variable levels readily detected arginine-methylated RUNX1 (using au- of PRMT1, RUNX1, and arginine-methylated RUNX1 toradiography) (data not shown). We also detected argi- proteins in different hematopoietic cell lineages, which nine methylation of RUNX1a at its C terminus (between further suggests that this post-translational modification amino acids 182 and 250) and within the Runt domain is involved in fine-tuning RUNX1 transcriptional regu- (within an “SGRGK” sequence that is also found at the latory activity.
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