Antagonistic actions of Rcor proteins regulate LSD1 activity and cellular differentiation Ghanshyam Upadhyaya, Asif H. Chowdhurya, Bharat Vaidyanathanb, David Kima, and Shireen Salequea,1 aDepartment of Biology, City College of New York and Graduate Center of City University of New York, New York, NY 10031; and bImmunology and Microbial Pathogenesis Program, Weill-Cornell Medical School and Sloan Kettering Institute, New York, NY 10021 Edited* by Stuart H. Orkin, Children’s Hospital and the Dana-Farber Cancer Institute, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA, and approved April 15, 2014 (received for review March 6, 2014) Lysine-specific demethylase 1 (LSD1) demethylates nucleosomal deletion diminishes or abolishes differentiation of normal he- histone H3 lysine 4 (H3K4) residues in collaboration with the co- matopoietic stem and progenitor cells (12, 13), respectively. In repressor CoREST/REST corepressor 1 (Rcor1) and regulates cell contrast, the Rcor1 germline deletion was recently demon- fates by epigenetically repressing gene targets. The balanced reg- strated to specifically abrogate erythroid differentiation (14). ulation of this demethylase, if any, is however unknown. We now Intriguingly, LSD1 is overexpressed in leukemic stem cells, demonstrate the actions of two other Rcor paralogs, Rcor2 and where it either sustains the oncogenic potential (15) or sup- Rcor3, in regulating LSD1 enzymatic activity and biological func- presses differentiation (16). These apparent anomalies may tion in hematopoietic cells. All three Rcor proteins interact with imply the existence of additional regulatory mechanisms for LSD1 and with the erythro-megakaryocytic transcription factor modulating LSD1 activity in specific cellular contexts. growth factor independence (Gfi)1b; however, whereas Rcor2, like Here we delineate the actions of two additional Rcor factors, Rcor1, facilitates LSD1-mediated nucleosomal demethylation, Rcor2 and Rcor3, in regulating LSD1 activity and function. We Rcor3 competitively inhibits this process. Appending the SANT2 further demonstrate the consequences of these processes on erythro-megakaryocytic differentiation and the actions of Rcor3 domain of Rcor1 to Rcor3 confers the ability to facilitate LSD1- on cognate gene targets. Finally, we correlate the effects medi- mediated demethylation on the chimeric Rcor protein. Consistent ated by these factors with their endogenous expression in pri- with their biochemical activities, endogenous Rcor1, Rcor2, and BIOLOGY mary hematopoietic cells to account for their in vivo functions. LSD1 promote differentiation, whereas Rcor3 opposes these pro- DEVELOPMENTAL cesses. Recruitment of Rcor3 to cognate gene targets by Gfi1b and Results LSD1 leads to inhibition of H3K4 demethylation of chromatin and LSD1 and Rcor1-3 were previously identified in a proteomic transcriptional derepression of these loci. Remarkably, profound screen for Gfi1b-associated proteins (8). Subsequent amino acid alterations in Rcor1/3 levels during erythroid versus megakaryo- sequence alignments of the Rcor proteins revealed conservation of cytic differentiation potentiate antagonistic outcomes. In mature most of the major domains (Elm2, SANT1, and linker) among erythroid cells, a strong upsurge in Rcor3 and a sharp decline in them (Fig. S1A). Consistent with the presence of a conserved Rcor1 levels counteract LSD1/Rcor1/2-mediated differentiation. In linker domain, Rcor2 and Rcor3, like Rcor1, associated with contrast, the opposite changes in Rcor1/3 levels in megakaryocytes LSD1 and with both Gfi1b and Gfi1 (Fig. 1A), but not with favor differentiation and likely maintain homeostasis between the SNAG domain mutant of Gfi1b (P2A-Gfi1b), thus reaffirming these lineages. Overall, our results identify Rcor3 as a natural in- the function of this domain in recruiting LSD1/Rcor factors to hibitor of LSD1 and highlight a dual mechanism of regulating the Gfi1/1b protein complexes (8, 17–19). The lack of interactions enzymatic activity and restraining the epigenetic impact of this with an unrelated protein, BirA (Fig. 1A, Bottom), and with total robust demethylase during hematopoietic differentiation. mouse IgG (Fig. S1 B and C) further confirmed the specificity of the interactions among these proteins. Subsequently, association ysine-specific demethylase 1 (LSD1; also known as Kdm1a, between the endogenous proteins in erythroid cells was con- firmed by coelution of Gfi1b/LSD1/Rcor protein complexes by LAof2, and BHC110) removes dimethyl or monomethyl moi- B C eties from histone H3 lysine 4 (H3K4) residues on free histones size-exclusion chromatography (gel filtration) (Fig. 1 and ). (1), but requires facilitation from the corepressor CoREST/ REST corepressor 1 (Rcor1) to catalyze this reaction on chro- Significance matin (nucleosomes) (2, 3). LSD1 also has been reported to demethylate H3K9 residues on chromatin in a context-dependent This work illustrates the dual regulation of the potent and manner, but not in vitro (4, 5). Various experiments have de- widely expressed demethylase lysine-specific demethylase 1 lineated the roles of distinct LSD1 and Rcor1 domains in medi- (LSD1) by the antagonistic actions of stimulatory and inhibitory ating interactions between them and/or in facilitating catalysis. Rcor factors and their consequences on hematopoietic dif- Whereas the ∼120-aa-long “linker” region of Rcor1 mediates in- ferentiation. Given the crucial role of LSD1 in diverse de- teraction with LSD1 (3), either both SANT (Swi3, Ada2, N-CoR, velopmental contexts, including ES cells, multiple mammalian and TFIIIB) domains (2) or only the SANT2 domain (3) have tissues, and oncogenesis, these results could potentially re- been identified as essential for facilitating catalysis. Biophysical flect the function of LSD1 and Rcor1-3 in multiple normal and studies subsequently revealed the “nanoscale clamp” formed by aberrant developmental contexts. Moreover, these insights the LSD1/Rcor1 co-complex on nucleosomes (6, 7) and high- could be used to develop targeted strategies for controlling lighted the contribution of the SANT2 domain in stabilizing this LSD1 activity in different diseases. structure. The contributions of the SANT or other domains to the cellular functions of Rcor1 remain unknown, however. Author contributions: S.S. designed research; G.U., A.H.C., B.V., and D.K. performed re- LSD1 and Rcor1 are recruited by different transcription search; S.S. contributed new reagents/analytic tools; G.U., B.V., and S.S. analyzed data; factors in diverse tissues and contexts. In hematopoietic cells, and S.S. wrote the paper. they associate with growth factor independence (Gfi)1 and The authors declare no conflict of interest. Gfi1b and repress the majority of Gfi1b gene targets in ery- *This Direct Submission article had a prearranged editor. throid cells (8). This duo also associates with Scl1/Tal1 (9, 10) 1To whom correspondence should be addressed. E-mail: [email protected]. and Bcl11A (11) in erythroid cells and mediates repression of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. their target genes. Therefore, LSD1 inhibition or conditional 1073/pnas.1404292111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1404292111 PNAS | June 3, 2014 | vol. 111 | no. 22 | 8071–8076 Downloaded by guest on September 24, 2021 ABthe addition of LSD1 and Rcor1-3 to mononucleosomes, attesting to the specificity of LSD1 activity in vitro (Fig. 2 A and C). Because the absence of the SANT2 domain is the major dis- tinguishing feature between Rcor3 and Rcor1/2, we appended each of the Rcor1 SANT domains to Rcor3 downstream of the linker region and evaluated the activity of the resulting chimeric proteins (Fig. 2 D and E). Strikingly, addition of the SANT2 C domain conferred the ability to facilitate LSD1 mediated nu- cleosomal demethylation on the chimeric Rcor3-SANT2 protein (Fig. 2D), but not that of the SANT1 domain (Fig. 2E). These results demonstrate inherent functional differences between the Rcor1 SANT domains even when compensated for positional differences in the Rcor backbone, and illustrate the structural basis for the inhibitory properties of Rcor3 relative to Rcor1/2. Fig. 1. Association of Rcor1-3 with LSD1 and Gfi proteins. (A) Western blot We next interrogated the function of the Rcor proteins in analysis of anti-myc immunoprecipitated material (IP: α-myc) alongside 10% modulating differentiation of the erythroid and megakaryocytic of input extract (Input) from cells expressing combinations of epitope-tag- lineages specified by Gfi1b (21). To do so, we knocked down LSD1 ged proteins. Shown are cellular extracts containing one of each type of and Rcor1-3 in primary hematopoietic cells (Fig. S3 and Fig. 3, epitope-tagged protein: myc (Rcor1, Rcor2, and Rcor3 in lanes 1, 2, and 3, respectively) and performed multiple assays to ascertain differenti- respectively), FLAG (LSD1), and V5 (Gfi1b-top, Gfi1-second from top, P2A- ation. Alterations in mRNA levels (Fig. 3 B and G), histochemical Gfi1b-third from top and BirA bottom panels) in different combinations staining (benzidine and acetylcholine esterase for erythroid and after immunoprecipitation and blotting as indicated. (B and C) Coelution of megakaryocytic cells, respectively) (Fig. 3 D and H), and surface endogenous Gfi1b, LSD1, and Rcor1 from uninduced MEL cells (B) and Gfi1b, protein expression (by FACS analysis) (Fig. 3 E and I)inthema- LSD1, and Rcor3 from induced MEL cells (C)