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Heterogeneous Nuclear Ribonucleoprotein C1 C2, Mecp1

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Heterogeneous Nuclear Ribonucleoprotein C1 C2, Mecp1 Heterogeneous nuclear ribonucleoprotein C1͞C2, MeCP1, and SWI͞SNF form a chromatin remodeling complex at the ␤-globin locus control region Milind C. Mahajan*, Geeta J. Narlikar†, Gokul Boyapaty*, Robert E. Kingston‡, and Sherman M. Weissman*§ *Department of Genetics, The Anlyan Center, Yale University School of Medicine, New Haven, CT 06511; †Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143; and ‡Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 Contributed by Sherman M. Weissman, August 31, 2005 Locus control regions (LCRs) are regulatory DNA sequences that are promoter (20–23). These studies suggest the possible association situated many kilobases away from their cognate promoters. LCRs of the LCR with specific chromatin remodeling activities, al- protect transgenes from position effect variegation and hetero- though such a LCR-specific chromatin remodeling activity has chromatinization and also promote copy-number dependence of not been isolated. In the present work, we describe the biochem- the levels of transgene expression. In this work, we describe the ical purification and properties of a previously undescribed biochemical purification of a previously undescribed LCR-associ- chromatin-remodeling complex that binds to the human ␤-globin ated remodeling complex (LARC) that consists of heterogeneous LCR HS2 in a sequence-specific manner. nuclear ribonucleoprotein C1͞C2, nucleosome remodeling SWI͞ SNF, and nucleosome remodeling and deacetylating (NuRD)͞ Materials and Methods MeCP1 as a single homogeneous complex. LARC binds to the Cell Culture and Preparation of Nuclear Extracts. Growth of the K562 hypersensitive 2 (HS2)-Maf recognition element (MARE) DNA in a cells and preparation of the nuclear extract is described in ref. 24. sequence-specific manner and remodels nucleosomes. Heteroge- For large-scale cultures, 2 liters of the K562 cells grown in neous nuclear ribonucleoprotein C1͞C2, previously known as a 175-cm flasks were transferred to the 10-liter Cytostir Cell general RNA binding protein, provides a sequence-specific DNA Culture jar and grown until the cell density reached 0.75 ϫ 106 recognition element for LARC, and the LARC DNA-recognition per ml. sequence is essential for the enhancement of transcription by HS2. Independently of the initiation of transcription, LARC becomes Electrophoretic Mobility Shift Assay (EMSA). The EMSA and tran- associated with ␤-like globin promoters. sient transfection assays were conducted as described in ref. 24. Unless otherwise indicated, the EMSA binding mixture con- NuRD͞MeCP1 ͉ HS2 tained 4 ng of 32P-labeled double-stranded oligonucleotide and 5 ␮g of crude k562 nuclear extract or 2 ␮g of K562 nuclear extract he human ␤-globin locus control region (LCR) is associated partially purified on a heparin-agarose column. Twith seven DNase hypersensitive (HS) sites situated Ϸ50 kb upstream from the 5Ј end of the adult ␤-globin gene (1, 2). Of Antibodies (Abs), Immunoprecipitation, and Western Blotting. The these seven HS sites, the first four, HS1, HS2, HS3, and HS4, are Abs against Brg1 (catalog no. SC-17796 and SC-12520x), erythroid specific (1). HS5 is present in several hematopoietic BAF155 (SC-10756x), actin (SC-10731), HDAC1 (SC-6298), lineages and is suggested to possess insulator activity (3–5), and INI1 (SC-13055x), MBD2 (SC-9397), MBD3 (SC-9402), Mi2␤ HS6 and HS7 are present in a variety of cell types. (SC-12541), and heterogeneous nuclear ribonucleoprotein HS2 acts as a classical enhancer in transient transfection assays (hnRNP) C1͞C2 (SC-15286) were purchased from Santa Cruz and in transgenic mice (6–9) and has binding sites for several Biotechnology. The Ab against MTA2 (PC656-1) was purchased transcription factors (10). A tandem pair of activator protein 1 from Oncogene Research (Cambridge, MA). The RbAP48 Ab (AP1)͞NF-E2 sites possesses the core enhancing activity of HS2 (catalog no. 05-524) was purchased from Upstate Biotechnology (11, 12). The AP1͞NF-E2 site is also called Maf recognition (Lake Placid, NY). Immunoprecipitation (IP) and immu- element (MARE). The erythroid-specific basic leucine zipper nodepletion procedures were carried out at 4°C. The LCR- (bZIP) protein p45 NF-E2 and several other members of this associated remodeling complex (LARC) eluted from the hepa- family, including NF-E2-related factor (NRF) 1, NRF2, Bach1, rin-agarose column was dialyzed against 10 mM Hepes buffer and Bach2, dimerize with the Maf proteins and bind to the (pH 7.9) containing 10% glycerol, 50 mM KCl, 1 mM MgCl2,1 NF-E2 site of the MARE sequence (2). However, p45 NF-E2, mM DTT, 1 mM PMSF, and Complete protease inhibitor tablet NRF1, and NRF2 knockout mice have normal erythropoiesis (at one tablet per 100-ml buffer concentration; Roche Diagnos- and globin expression (2), suggesting that MARE has an addi- tics), concentrated to Ϸ1mg͞ml, and used for IP and immu- tional role beyond the binding of NF-E2 and Maf family of nodepletions. For each IP, 500 ␮l of the K562 nuclear protein proteins. containing Ϸ500 ␮g of the protein and 3 ␮g of each of the Many deletion and transgenesis studies have been performed anti-Brg1, Mi2␤, MTA2, and hnRNP C1͞C2 Ab was used. Three with the ␤-globin LCR (1, 13) that suggest the importance of HS2 for transactivation of ␤-like globin genes and for the formation of DNase HS sites (7, 14–16). However, deletion of Abbreviations: AP1, activator protein 1; hnRNP, heterogeneous nuclear ribonucleoprotein; the LCR DNA sequences severely reduces the transcription of HS, hypersensitive; IP, immunoprecipitation; LC-MS͞MS, liquid chromatography-tandem the ␤-like genes, but the DNase hypersensitivity and polymerase MS; LCR, locus control region; LARC, LCR-associated remodeling complex; MARE, Maf ␤ recognition element; NuRD, nucleosome remodeling and deacetylating; PCS, promoter II association with promoters of the remaining -globin genes conserved sequence. persists (17–19). Several biochemical studies in cell lines suggest §To whom correspondence should be addressed at: Department of Genetics, The Anlyan that the MARE sequences of HS2, apart from their involvement Center, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06510. E-mail: in the enhancement of transcription, also take part in the [email protected]. alteration of the chromatin structure of HS2 and a linked © 2005 by The National Academy of Sciences of the USA 15012–15017 ͉ PNAS ͉ October 18, 2005 ͉ vol. 102 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507596102 Downloaded by guest on September 28, 2021 micrograms of the goat and rabbit IgG were added to the K562 nuclear proteins in a control experiment. Western blotting was performed as described in ref. 24. Five hundred micrograms of K562 nuclear extract, eluted with 0.6 M NaCl from heparin- agarose column, was immunodepleted with 70 ␮g each of Abs against Brg1, Mi2␤, and hnRNP C1͞C2. A mixture of 35 ␮g each of normal rabbit and goat IgG was used as control. The Abs and K562 extracts were incubated overnight followed by 2-h incu- bation with 100 ␮l of Protein-G beads. The beads were separated by centrifugation, and the supernatants were analyzed by West- ern blotting using Abs against several LARC components. Purification of the LARC as a MARE Binding Activity. Throughout the purification procedure, the LARC was assayed by EMSA as a MARE binding activity. The entire purification procedure was carried out at 4°C, and, unless otherwise indicated, all of the buffers contained 1 mM PMSF, 1 mM bestatin, 1 mM diisopro- pyl fluorophosphate, 1 ␮g͞ml L-1-tosylamido-2-phenylethyl Fig. 1. Some prominent non-NF-E2-containing proteins interact with the chloromethyl ketone, and 7-amino-1-chloro-3-tosylamido-2- MARE sequence of HS2. (A) A schematic diagram showing the ␤-globin locus heptanone, and Complete protease inhibitor pill (Roche) per 50 and the position of the MARE sequence at the HS2 site of the LCR. (B) Western ml of buffer. Typically, the starting K562 nuclear extract isolated blot of the K562 nuclear extract fractionated on the heparin-agarose column. from 8–10 ϫ 109 K562 cells containing 85–120 mg of protein in Sixty-five micrograms each of the crude K562 nuclear extract and the extracts a nuclear extract storage buffer (20 mM Hepes, pH 7.9͞100 mM sequentially eluted from the heparin-agarose column with 0.42 and 0.6 M ͞ ͞ ͞ NaCl were subjected to Western analysis with Ab against p45 NF-E2. (C) EMSAs KCl 1 mM MgCl2 20% glycerol 1 mM DTT) was loaded onto with 0.6 M NaCl heparin-agarose fraction of the K562 nuclear extracts and a 30-ml heparin-agarose column preequilibrated with the same double-stranded MARE sequence and its mutant variants. Sequences of the buffer. The column was washed with 300 ml of the storage buffer positive strands of the double-stranded MARE oligonucleotide and its mu- followed by 300 ml of 0.2 M NaCl and 300 ml of 0.42 M NaCl in tants are shown. 10 mM Hepes buffer (pH 7.9) containing 10% glycerol, 1 mM MgCl2, and 1 mM DTT. The MARE-binding activity was eluted from the column by 0.6 M NaCl in the same buffer. The active 0.6 M NaCl did not contain the p45 NF-E2, but these proteins fractions were detected by EMSA, pooled, and dialyzed against were able to bind to the MARE sequence (Fig. 1C). We have the DNA-affinity column buffer consisting of 10 mM Hepes previously shown these bands to be sequence specific (24). buffer (pH 7.9) containing 10% glycerol, 50 mM KCl, 1 mM Mutation in the NF-E2 binding site did not disturb these EMSA MgCl2, and 1 mM DTT. After dialysis, the protein was centri- bands. However, mutations in the second AP1 sequence at the fuged at 15,000 rpm in a Sorvall SS-34 rotor for 15 min and the 3Ј end of the NF-E2 sequence (Mut1 and Mut2) neutralize these clear supernatant was collected.
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