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. 2005 Oct 18;102(42):15012-7.
doi: 10.1073/pnas.0507596102. Epub 2005 Oct 10.

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the beta-globin locus control region

Milind C Mahajan  1 , Geeta J Narlikar, Gokul Boyapaty, Robert E Kingston, Sherman M Weissman
Affiliations

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the beta-globin locus control region

Milind C Mahajan et al. Proc Natl Acad Sci U S A. .

Abstract

Locus control regions (LCRs) are regulatory DNA sequences that are situated many kilobases away from their cognate promoters. LCRs protect transgenes from position effect variegation and heterochromatinization and also promote copy-number dependence of the levels of transgene expression. In this work, we describe the biochemical purification of a previously undescribed LCR-associated remodeling complex (LARC) that consists of heterogeneous nuclear ribonucleoprotein C1/C2, nucleosome remodeling SWI/SNF, and nucleosome remodeling and deacetylating (NuRD)/MeCP1 as a single homogeneous complex. LARC binds to the hypersensitive 2 (HS2)-Maf recognition element (MARE) DNA in a sequence-specific manner and remodels nucleosomes. Heterogeneous nuclear ribonucleoprotein C1/C2, previously known as a general RNA binding protein, provides a sequence-specific DNA recognition element for LARC, and the LARC DNA-recognition sequence is essential for the enhancement of transcription by HS2. Independently of the initiation of transcription, LARC becomes associated with beta-like globin promoters.

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Figures

Fig. 1.
Fig. 1.
Some prominent non-NF-E2-containing proteins interact with the MARE sequence of HS2. (A) A schematic diagram showing the β-globin locus and the position of the MARE sequence at the HS2 site of the LCR. (B) Western blot of the K562 nuclear extract fractionated on the heparin-agarose column. Sixty-five micrograms each of the crude K562 nuclear extract and the extracts 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 with 0.6 M NaCl heparin-agarose fraction of the K562 nuclear extracts and double-stranded MARE sequence and its mutant variants. Sequences of the positive strands of the double-stranded MARE oligonucleotide and its mutants are shown.
Fig. 2.
Fig. 2.
Purification and characterization of the LARC of the β-globin LCR. (A) Schematic diagram of the protocol used for the biochemical purification of the LARC. The total protein obtained at each purification step of a typical purification procedure is mentioned in brackets. (B) EMSA with the 32P-labeled MARE oligonucleotide sequence and the K562 nuclear extracts at different purification stages. The “Free Oligo” lane is the EMSA without the K562 nuclear extract. (C) Display of the purified LARC on a 10% SDS/PAGE gel. The gel bands were digested with modified trypsin (Sigma), and their protein compositions were identified by MALDI and LC-MS/MS analysis. The asterisk-marked bands are the degradation products of the high-molecular-mass bands. They inconsistently appear on the gel. BAF 170, BAF 155, and BAF 53 were identified by MALDI, and the rest of the proteins, except MBD2 and MBD3, were identified by LC-MS/MS analysis. (D) Western blots of 4–8 μg of purified LARC with Abs against hnRNP C1/C2 and some components of SWI/SNF and NuRD complexes.
Fig. 3.
Fig. 3.
The SWI/SNF, NuRD/MeCP1, and hnRNP C1/C2 interact with the MARE sequence of HS2 as a single homogeneous complex. (A) EMSA of the MARE oligonucleotide with the Superose-6 column fractions of K562 nuclear extracts. Elution peaks of the molecular-mass standards are marked at the top. (B) Western blots of the 30 μl each of Superose-6 fractions with Abs against Brg1, Mi2β, MTA2, RbAP48, INI1, and hnRNP C1/C2. (C) Immunoprecipitations and immunodepletions of the K562 nuclear extracts from the heparin-agarose column. (i) Immunoprecipitates obtained with the Abs against the Mi2β, Brg1, and hnRNP C1/C2 were probed on a Western blot with Abs against Brg1, Mi2β, BAF155, and hnRNP C1/C2. (ii) The K562 nuclear extracts fractionated on the heparin-agarose column with 0.6 M NaCl were immunodepleted with Abs against Mi2β, Brg1, and hnRNP C1/C2 and analyzed by Western blotting with Abs against nine proteins as shown on the left-hand side. (D) Electrophoretic mobility supershift/neutralization assay of the LARC–EMSA bands. Crude K562 nuclear extracts and 32P-labeled MARE oligonucleotide were used for the gel supershift assays with 2 and 5 μg of the Abs against Mi2β and BAF155, respectively.
Fig. 4.
Fig. 4.
Role of hnRNP C1/C2 in binding of LARC to its target MARE–DNA. (A) Southwestern hybridization of the purified LARC complex. Approximately 10 μg of the LARC complex was run in three lanes on a 4–15% SDS/PAGE with standard molecular-mass markers and blotted on a Millipore Immobilon-P membrane. Each lane on the blot was cut and processed separately. Blots i and ii were probed with 100 ng each of 32P-labeled MARE and Mut2 oligonucleotides (see Fig. 1), respectively. The third blot (iii) was analyzed by Western using hnRNP C1/C2 Ab. The Southwestern blot (i) was stripped off its radioactivity and analyzed (iv) by using hnRNP C1/C2 Ab. (B) Western blots of UV-crosslinked LARC. Approximately 5 μg of purified LARC was UV-crosslinked with the BrdUrd-containing MARE oligonucleotide and analyzed by using Abs against hnRNP C1/C2 and MTA2 as indicated in the figure. Lane 1, UV-crosslinked LARC in the presence of BrdUrd containing MARE oligonucleotide; lane 2, UV-crosslinked LARC in the absence of the MARE–DNA. (C) Gel supershift assay of the LARC–EMSA bands in the presence of the anti-hnRNP C1/C2 Ab. The EMSA was performed with crude K562 nuclear extract, 32P-labeled MARE DNA, and 2 μg each of hnRNP C1/C2 Ab and rabbit IgG.
Fig. 5.
Fig. 5.
Nucleosome remodeling activity of the purified LARC. (A) Restriction enzyme accessibility assay to show the remodeling of the dinucleosome constructed with a 375-bp HS2 by LARC. The position of the PstI restriction enzyme site on the first nucleosome situated at the 5′ end of the MARE sequence is indicated by an arrow (see also Table 1, which is published as supporting information on the PNAS web site). The gel picture shows the time course of Pst-1 cutting of the nucleosomal DNA in the presence and absence of ATP. Comparison of remodeling of WT and mutant HS2 (Mut2) containing dinucleosomes is made in the bar diagrams. WT and Mut2 MARE sequences are described in Fig. 1. (B) Remodeling of a trinucleosome containing 490-bp DNA consisting of the 375-bp HS2, PstI, and BglII restriction enzyme sites at the 5′ and 3′ ends, respectively, and multimer of the LARC binding sequence with a HhaI site in the middle (Table 1). The bar diagram shows the relative accessibility of the three restriction enzyme sites in the presence of the LARC and a SWI/SNF complex from the HeLa cells.
Fig. 6.
Fig. 6.
In vivo and in vitro analysis of the DNA-binding properties of the LARC to the β-like promoters and the LCR. (A) ChIP assays of formaldehyde-fixed <500 bp K562 chromatin with the Abs against Brg1, Mi2β, MTA2, and hnRNP C1/C2. The sequences of the PCR primers are listed in Table 1. This figure is a representative of three separate ChIP experiments. (B–D) Each EMSA binding mixture contained 2 ng of the double-stranded oligonucleotide (≈100,000 cpm) and 2 μg of the partially purified LARC from heparin-agarose column. The sequences of the oligonucleotides are given in Table 1. (B) Comparative EMSA of the 32P-labeled MARE sequence and the oligonucleotides from the γ-globin promoter spanning a 286-bp sequence immediately upstream from the transcription start site. Free probe lane contains a mixture of all of the oligonucleotides without the K562 nuclear extract. (C) The competitive EMSA of the 32P-labeled MARE sequence with 100-fold molar excess of the nonradioactive oligonucleotides from the γ-globin promoter. (D) Comparative EMSAs of the 32P-labeled MARE and the β-PCS oligonucleotides and their competitive binding in the presence of the 100-fold molar excess of the nonradioactive counterparts.
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