Functional selectivity of recombinant mammalian SWI/SNF subunits

Abstract

The SWI/SNF family of chromatin-remodeling complexes plays a key role in facilitating the binding of specific transcription factors to nucleosomal DNA in diverse organisms from yeast to man. Yet the process by which SWI/SNF and other chromatin-remodeling complexes activate specific subsets of genes is poorly understood. We show that mammalian SWI/SNF regulates transcription from chromatin-assembled genes in a factor-specific manner in vitro. The DNA-binding domains (DBDs) of several zinc finger proteins, including EKLF, interact directly with SWI/SNF to generate DNase I hypersensitivity within the chromatin-assembled beta-globin promoter. Interestingly, we find that two SWI/SNF subunits (BRG1 and BAF155) are necessary and sufficient for targeted chromatin remodeling and transcriptional activation by EKLF in vitro. Remodeling is achieved with only the BRG1-BAF155 minimal complex and the EKLF zinc finger DBD, whereas transcription requires, in addition, an activation domain. In contrast, the BRG1-BAF155 complex does not interact or function with two unrelated transcription factors, TFE3 and NF-kappaB. We conclude that specific domains of certain transcription factors differentially target SWI/SNF complexes to chromatin in a gene-selective manner and that individual SWI/SNF subunits play unique roles in transcription factor-directed nucleosome remodeling.

Figure 1

Mammalian SWI/SNF selectively functions with several zinc finger DNA-binding proteins to remodel chromatin and activate transcription in vitro. (A) In vitro transcription of chromatin-assembled β-globin plasmids by zinc finger containing transcription factors (EKLF, GATA-1, and Sp-1) in the presence of mammalian SWI/SNF chromatin-remodeling complex. β-globin plasmid templates were assembled into chromatin and incubated in the absence (lanes 1–3, 6–8, 12–14) or presence (lanes 4,5, 9–11,15–17) of SWI/SNF and, where indicated, 37 pmole of recombinant EKLF (lanes 3,5); 30 nmole, 60 nmole, and 150 nmole of an Sp1 fraction (lanes 6–11), and 35 pmole, 45 pmole, or 65 pmole of recombinant GATA-1 (lanes 12–17) per 1 μg of chromatin in a 100 μL of reaction volume. Triangles indicate increasing concentrations of Sp1 (which was inhibitory to transcription at high levels in the absence of SWI/SNF) or GATA-1. All proteins were added to assembled chromatin and incubated for 30 min at 27°C. Chromatin assembly and transcription reactions were conducted as described in Materials and Methods. Primer extension products of the β-globin promoter and the AdLuc internal control gene are indicated by arrows. (B) Analysis of the ability of different zinc finger-containing DNA-binding proteins to generate DNase I hypersensitivity within the β-globin promoter in the presence of SWI/SNF. Assembled chromatin was incubated with SWI/SNF and either EKLF, Sp1, or GATA-1 as in A, and half of the reaction was divided in two and digested with 2 U and 1 U of DNase I as described in Materials and Methods. Triangles indicate increasing amounts of DNase I. Brackets show the −120–+10 region of the β-globin promoter. A schematic diagram of the β-globin promoter is shown between panels A and B.

Figure 2

Distinct protein domains of EKLF are required for SWI/SNF-dependent chromatin remodeling and transcriptional activation. (A) Analysis of different EKLF mutant proteins in β-globin promoter activation in the presence or absence of SWI/SNF in vitro. Assembled chromatin templates were incubated with either wild-type or mutant EKLF proteins (37 pmole/1 μg of chromatin in a 100 μL reaction volume) and SWI/SNF, as indicated for each lane. The reactions were then split, and half was transcribed as in Figure 1A and as described in Materials and Methods. (B) The zinc finger DNA-binding domain of EKLF is sufficient to direct SWI/SNF-dependent DNase I hypersensitivity within the β-globin promoter. After assembly, chromatin was incubated with either wild-type or mutant EKLF protein in the presence or absence of SWI/SNF, as indicated above each lane. Reactions were then split and half was transcribed as shown in A and the remaining chromatin was divided into two tubes with 150 ng chromatin per tube and digested with 1 U and 2 U of DNase I. Triangles indicate increasing amounts of DNase I. Brackets show the −120–+10 region of the promoter. (M) Digested β-globin plasmid as a size marker. Bands represent digests of NcoI (4.7 Kb), NcoI/BsaAI (841 bp) and NcoI/EcoRI (301 bp). A schematic diagram of the domain structure of EKLF is shown between panels A and B.

Figure 3

SWI/SNF subunits interact specifically with the zinc finger DNA-binding domains (DBDs) of EKLF, GATA-1, and Sp1. (A) Mammalian SWI/SNF interacts with zinc finger DBDs. GST pull-down assays were performed with 3 μg SWI/SNF and 1 μg GST-fused wild-type or mutant EKLF, GATA-1, TFE3, NF-κB (p50). SWI/SNF subunits were detected by Western blot analyses using the antisera indicated on the left as described in Materials and Methods. (B) Acidic and proline-rich activation domains do not interact with mammalian SWI/SNF. GST pull-down assays were performed using 1.5 μg of SWI/SNF and 500 ng each of GST-fused wild-type or mutant EKLF and VP16 proteins. Histidine pull-down assays were performed using 500 ng each of wild-type EKLF, EKLF DBD, wild-type GAL4–VP16, and GAL4 DBD as described in Materials and Methods. Proteins that were pulled down with the beads were analyzed on a 10% SDS-PAGE gel and immunoblotted with antibodies against SWI/SNF subunits, BRG1, BAF170, BAF155, and BAF57. (C) Specific SWI/SNF subunits interact with the EKLF zinc finger DBD. (Top panel) Flag-tagged hSWI/SNF was analyzed by SDS-PAGE, stained with silver (lane 4), blotted onto a PVDF membrane, and processed for far-Western analysis using three different probes: GST–EKLF, followed by anti-EKLF antibody (lane 2), ³²P-labeled GST–EKLF (lane 1), or GST–EKLF (AD) followed by anti-EKLF antibody (lane 3) as described in Materials and Methods. The positions of SWI/SNF subunits, BRG1, BAF170, and BAF155 are indicated. (Bottom panel) Wild-type EKLF and the EKLF DBD interact with recombinant SWI/SNF subunits. GST pull-down assays were carried out using 200 ng of purified recombinant F-BRG1, F-BAF170, or F-BAF155 with 200 ng of GST-fused wild-type or mutant EKLF and Sp1 proteins. Equal amounts of supernatants (S) and beads (B) were analyzed on a 10% SDS-PAGE gel and immunoblotted with antibodies against SWI/SNF subunits, BRG1, BAF170, and BAF155.

Figure 4

A minimal recombinant SWI/SNF complex is sufficient for EKLF-dependent transcriptional activation of chromatin-assembled β-globin genes. Recombinant BRG1 and BAF155 or BAF170 cooperate with EKLF to activate chromatin-assembled β-globin genes in vitro. After nucleosome assembly, 100 ng of chromatin was incubated with: 3.7 pmole of EKLF as indicated, 20 ng of F-BRG1 (lanes 4,5,10–16,18–19,21–22), 140 ng of F-BAF170 (lanes 8–9,14–15,17–19), 100 ng of F-BAF155 (lanes 10–11), and 140 ng of F-BAF155 (lanes 12–15,20–22), followed by primer extension analysis of transcripts. As a positive control, an aliquot of 58 ng purified SWI/SNF was incubated with 3.7 pmole of EKLF (lane 3).

Figure 5

Recombinant SWI/SNF subunits do not support transcription from chromatin-assembled HIV-1 promoters by TFE3 and NF-κB. Factor-dependent transcriptional activation by native and recombinant SWI/SNF on a chromatin-assembled HIV-1 promoter. (Left ) an aliquot of 100 ng pHIV-1–Luc was assembled into chromatin in the absence of transcription factors (lanes 1–5), or with the following recombinant proteins: 4 pmole EKLF (lanes 6–8) or 5 pmole TFE3 plus one pmole NF-κB subunits (p50:p65; lanes 9–13). Where indicated, 20 ng F-BRG1, 140 ng F-BAF155, and 58 ng native SWI/SNF were added after nucleosome assembly. (Right) A mixture of 5 pmole TFE3 and 1 pmole NF-κB (p50:p65) was incubated with 100 ng HIV-1 chromatin either before (lanes 16–17) or after (lanes 18–19) nucleosome assembly. Transcription reactions contained (lanes 15,17,19) or lacked (lanes 14,16,18) 58 ng native SWI/SNF. The α-globin gene was included as a control for transcription and RNA recovery. A schematic diagram of the HIV-1 promoter is shown below.

Figure 6

The EKLF zinc finger DNA-binding domain (DBD) is sufficient to target chromatin remodeling by the BRG1–BAF155 minimal complex. Analysis of recombinant SWI/SNF subunits to generate DNase I hypersensitive sites within the chromatin-assembled β-globin promoter in the presence of the EKLF DBD. After nucleosome assembly, 300 ng of chromatin was incubated with the following proteins as indicated: recombinant SWI/SNF subunits (60 ng F-BRG1, 400 ng F-BAF155, 400 ng F-BAF170); native SWI/SNF (180 ng); and EKLF DBD (11 pmole). 100 ng of the reaction was transcribed as a control (data not shown) and the remaining chromatin was divided in half and digested with 2 U and 1 U of DNase I as described in Materials and Methods. Triangles indicate increasing amounts of DNase I. (M) Digested β-globin plasmid used as a size marker. Bands represent digests of NcoI (4.7 kb), NcoI/BsaAI (841 bp) and NcoI/EcoRI (301 bp).

Figure 7

Interaction of recombinant SWI/SNF subunits with the EKLF DNA-binding domain (DBD) and DNA. A 60-bp region of the β-globin promoter containing the EKLF binding site was incubated with the following proteins: 10 ng of EKLF DBD (lanes 2,8–15); 20 ng F-BRG1 (lanes 3,6–7,11–15); 140 ng of F-BAF155 (lanes 4,6,9,12,14); 140 ng of F-BAF170 (lanes 5,7,10,13,15) and analyzed by EMSA. One microliter of undiluted antibodies was added as indicated.

Figure 8

Model for targeted chromatin remodeling and transcriptional activation, highlighting the interaction between the EKLF zinc finger DNA-binding domain and mammalian SWI/SNF subunits.