Volume 19, Issue 6, Pages 857-864 (September 2005) Regulation of LSD1 Histone Demethylase Activity by Its Associated Factors  Yu-Jiang Shi, Caitlin Matson, Fei Lan, Shigeki Iwase, Tadashi Baba, Yang Shi  Molecular Cell  Volume 19, Issue 6, Pages 857-864 (September 2005) DOI: 10.1016/j.molcel.2005.08.027 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 CoREST Regulates LSD1-Mediated Histone Demethylation (A) LSD1 purified from HeLa cells but not recombinant LSD1 can demethylate nucleosomal substrates. Free histones (Bulk) or mononucleosomes (Nuc) were used as substrates for demethylation reactions catalyzed by either recombinant His-LSD1 purified from bacteria or LSD1 complex purified from HeLa cells. The demethylation reaction was analyzed by Western blotting using an anti-dimeK4H3 or anti-dimeK9H3 control antibody. (B) Identification of LSD1-associated proteins. The LSD1-associated factors were purified using tandem tag affinity purification (Shi et al., 2003), separated on a 4%–12% gradient SDS NUPAGE gel, and visualized by silver staining. The identities of these proteins were revealed by MS/MS and indicated on the right. (C–E) Reconstitution of the LSD1 demethylase activity in vitro. Demethylation of nucleosomal histones by His-LSD1 was carried out in the presence of either recombinant CoREST (C), HDAC1 (D), or BHC80 (E), and the demethylation reactions were analyzed by Western blotting as above. (F) Comparable demethylation of free histones by recombinant LSD1 in the presence and absence of CoREST. Recombinant His-LSD1 demethylated free histones at similar efficiencies with (lane 4) or without CoREST (lane 2). (G) Demethylation of nucleosomal histones isolated from HeLa cells with or without TSA treatment. Mononucleosomes isolated from HeLa cells with (lanes 3 and 4) or without TSA treatment (lanes 1 and 2) were used for demethylation reactions by His-LSD1 in the presence (lanes 2 and 4) or absence of CoREST (lanes 1 and 3). Global histone hyperacetylation induced by TSA is shown in the bottom panel (compare lanes 3 and 4 with lanes 1 and 2) using a pan H3 acetylation-specific antibody and quantified on a bar graph. (H) TSA treatment causes derepression of LSD1 target genes. Total RNAs isolated from untreated (lane 1), TSA-treated (lane 2), and mock-treated (DMSO, lane 3) HeLa cells were subjected to RT-PCR to examine the expression levels of the LSD1 target genes SCN2A (top panel) and SCN3A (middle panel). β-actin (low panel) was used as a loading control. Molecular Cell 2005 19, 857-864DOI: (10.1016/j.molcel.2005.08.027) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Majority of the CoREST Stimulatory Activity toward LSD1-Mediated Demethylation of Nucleosomal Substrates Resides in the C-Terminal Region of CoREST (A) Diagrams of CoREST deletion mutants. (B) Analysis of the CoREST deletion mutants identifies the C-terminal region critical for stimulating LSD1 demethylase activity. The top panel represents Western blot analysis of LSD1 demethylase activity in the presence of wild-type or mutant CoREST proteins. The relative LSD1 demethylase activity was calculated by comparing with the H3-K9 dimethylation signals (middle panel) and is expressed in a bar graph (lower panel). Buffer without LSD1 (lane 1), showing no demethylase activity, was arbitrarily designated as 1. (C) Identification of a putative LSD1 interacting domain on CoREST. Comparable amounts of wt (wt) and deletion mutants of CoREST (Del 1–5) were incubated with either GST or GST-LSD1. Twenty percent of the input of the CoREST proteins is shown in the top panel. The bound proteins (middle panel) and the flow throughs (lower panel) were analyzed by SDS-PAGE and Coomassie blue staining. The asterisks (*) in the middle panel indicate the bound CoREST or CoREST mutants, and the arrowheads indicate the amount of GST or GSTLSD1 used for pull-down in each assay. The hand signs indicate unbound CoREST (lane 1) or CoREST mutants (lanes 3, 6, and 7) after incubation with either GST or GSTLSD1 beads. Molecular Cell 2005 19, 857-864DOI: (10.1016/j.molcel.2005.08.027) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Regulation of Endogenous LSD1 Stability by CoREST (A) Immunostaining of CoREST shRNA-transfected cells with CoREST and LSD1 antibodies. A GFP-expressing plasmid was cotransfected with a CoREST shRNA (small hairpin RNA) to mark the transfected cells (the left column). The cells were then stained with anti-CoREST (Ab), anti-LSD1 (Ae), and anti-DAPI antibodies, which mark the nucleus (Ac and Af). The GFP-positive cells in which the intended gene was inhibited by the corresponding CoREST shRNA are circled by dotted lines. Importantly, the CoREST shRNA reduced not only CoREST but also LSD1 levels. (B) Western blotting analysis of stable cell lines expressing shRNAs directed against LSD1, mutant GFP, CtBP, and CoREST. Extracts were prepared from these cells and blotted with antibodies indicated on the right. Actin was included as a loading control (bottom panel). (C) CoREST shRNA does not affect LSD1 mRNA level. RNAs were isolated from cells expressing shRNAs for CoREST, LSD1, and CtBP1 and were analyzed for CoREST and LSD1 expression by RT-PCR. Note that the CoREST shRNA did not alter LSD1 mRNA level (middle panel, compare lanes 1 and 2 with lanes 5 and 6). Note that ZL3VS treatment also did not change CoREST or LSD1 mRNA levels. (D) Proteasome inhibitor ZL3VS rescues LSD1 protein expression in cells expressing CoREST shRNA. The same stable cell lines in (B) were either not treated (lanes 1, 3, 5) or treated (lanes 2, 4, 6) with the proteasome inhibitor ZL3VS for 24 hr before Western blotting using antibodies indicated on the right. The LSD1 protein level was significantly increased in the CoREST shRNA-expressing cells after ZL3VS treatment (compare lanes 1 and 2, second panel). (E) Analysis of LSD1/CoREST target gene expression in stable cell lines expressing shRNA for LSD1, mGFP, and CoREST. RNAs were isolated from stable cells expressing the indicated shRNA, and the expression of LSD1 target genes SCN2A and SCN3A was analyzed by RT-PCR. Note derepression of the target genes in both the LSD1 (lane 2) and CoREST (lane 4) knockdown cells. (F) Analysis of LSD1 promoter occupancy and histone methylation status in the LSD1 and CoREST knockdown cell lines. LSD1 occupancy at the SCN2A and 3A promoters (+1 to −2 kb) was determined by chromatin immunoprecipitation (ChIP) in the mGFP-, LSD1-, and CoREST shRNA-expressing HeLa cells. Knockdown of either LSD1 or CoREST reduced promoter occupancy of both proteins as well as diMeK9H3 levels at the promoters. In contrast, H3-K4 methylation status was increased, as shown by ChIP using an anti-diMeK4H3 antibody. The input and IgG controls are indicated on the right. Molecular Cell 2005 19, 857-864DOI: (10.1016/j.molcel.2005.08.027) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 BHC80 Inhibits LSD1 Demethylase Activity (A) BHC80 inhibits LSD1 demethylase activity on free histones. The amounts of recombinant LSD1 and BHC80 are indicated on the top. The demethylation reactions were analyzed by Western blotting using antibodies indicated on the right. (B) BHC80 inhibition of LSD1-mediated demethylation is unaffected by CoREST. His-LSD1 was incubated either with buffer (lane 3), BHC80 +CoREST (lanes 2 and 4), or CoREST alone (lane 5) for 5 min. Then 2 μg of bulk histones was added to the reactions to start the demethylation reaction (lanes 1–5). Arrows indicate the positions of LSD1, CoREST, and BHC80. (C) Dose-dependent inhibition of CoREST-stimulated LSD1 demethylation by BHC80. His-LSD1was incubated with buffer (lane 2), CoREST (lanes 3 and 5–8), and different amounts of His-BHC80 ranging from 0 to 2 μg (lanes 5–8) for 5 min, and then 2 μg of nucleosomal histones was added to the demethylation reaction. With the increasing amounts of the BHC80 protein, a corresponding gradual decrease of demethylation reaction mediated by LSD1/CoREST is evident (lanes 5–8). Molecular Cell 2005 19, 857-864DOI: (10.1016/j.molcel.2005.08.027) Copyright © 2005 Elsevier Inc. Terms and Conditions