The activity of mammalian brm/SNF2alpha is dependent on a high-mobility-group protein I/Y-like DNA binding domain

Abstract

The mammalian SWI-SNF complex is a chromatin-remodelling machinery involved in the modulation of gene expression. Its activity relies on two closely related ATPases known as brm/SNF2alpha and BRG-1/SNF2beta. These two proteins can cooperate with nuclear receptors for transcriptional activation. In addition, they are involved in the control of cell proliferation, most probably by facilitating p105(Rb) repression of E2F transcriptional activity. In the present study, we have examined the ability of various brm/SNF2alpha deletion mutants to reverse the transformed phenotype of ras-transformed fibroblasts. Deletions within the p105(Rb) LXCXE binding motif or the conserved bromodomain had only a moderate effect. On the other hand, a 49-amino-acid segment, rich in lysines and arginines and located immediately downstream of the p105(Rb) interaction domain, appeared to be essential in this assay. This region was also required for cooperation of brm/SNF2alpha with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in high-mobility-group protein I/Y DNA binding domains and is required for the tethering of brm/SNF2alpha to chromatin.

FIG. 1

The C-terminal region of brm is required for reversion of the ras-induced transformed phenotype of DT cells. (A) Schematic representation of the various hbrm-derived constructs expressed in DT cells. The solid box represents the HA tag. (B and C) Extracts from DT-derived cell lines expressing either ΔE7 (clones E4 and E7) (B) or ΔCter(1337) (clones C15, C27, and C37) (C) were resolved by SDS-PAGE and analyzed by Western blotting with anti-hbrm antibodies. To allow comparison, panels B and C show levels of brm in the parental DT cells as well as in DT21 and A2 cells, which express WT hbrm and ATPmut, respectively. (D) Growth in soft agar. Parental DT cells or DT cells expressing either WT hbrm, ATPmut, ΔE7, or ΔCter(1337) were plated in 60-mm dishes (10² or 10³ cells per dish). The total number of visible colonies was scored after 15 days in culture and compared to the total number of cells initially seeded (plating efficiency, expressed as a percentage). The results shown here are averages and standard deviations from three independent experiments.

FIG. 2

Reversion of the ras-transformed phenotype by exogenous hbrm is dependent on the KR region. (A) Schematic representations of the C-terminal region of either WT hbrm (line 1), ΔKR (line 2), or ΔBromo (line 3). In all constructs, the N-terminal region that is not shown is WT. (B) Extracts from DT-derived cell lines expressing either ΔBromo or ΔKR were resolved by SDS-PAGE and analyzed by Western blotting with anti-hbrm antibodies. To allow comparison, expression levels in NIH 3T3 cells as well as in A2, E4, and C15 cells, expressing ATPmut, ΔE7, and ΔCter(1337), respectively, are also shown. (C) Growth in soft agar. Parental DT cells or DT cells expressing either WT hbrm, ΔBromo, or ΔKR were plated in 60-mm dishes (10² or 10³ cells per dish). The total number of visible colonies was scored after 15 days in culture and compared to the total number of cells initially seeded (plating efficiency, expressed as a percentage). The results shown here are averages and standard deviations from three independent experiments.

FIG. 3

The KR region is required for transcriptional synergy between hbrm and GR in the context of chromatin. A C33A-derived cell line containing an integrated MMTV CAT reporter construct was transfected with the vector without the insert (line 1) or with the GR expression vector either in the absence (line 2) or in the presence of an expression vector for WT hbrm (line 3), ATPmut (line 4), ΔCter(1337) (line 5), or ΔCter(1393) (line 6). The cells were harvested 36 h posttransfection, and CAT activity was measured. The results are shown as fold activation above CAT activity obtained with the vector without the insert and are compiled from seven independent experiments.

FIG. 4

Potential NLS and HMGI-like DNA binding domain within the KR region. (A) Amino acid sequence of hbrm between positions 1336 and 1384 of the published sequence. The putative bipartite NLS is underlined. The conserved motif found in the HMGI DNA binding domain is boxed. (B) Alignment of the putative NLS present in the KR region with the well-characterized NLS sequences from nucleoplasmin and N1 proteins. (C) Alignment of the KR region of hbrm with several proteins known or predicted to contain an HMGI-like DNA binding domain, also known as an AT hook.

FIG. 5

The KR region mediates hbrm DNA binding. (A) Schematic of the GST-hbrm fusion proteins used to assay the DNA binding properties of the hbrm KR region. (B) EMSA was performed with a 337-bp randomly chosen fragment of genomic Drosophila DNA and either GST alone (lane 1), wild-type GST-hbrm (lane 2), GST-ΔE7 (lane 3), GST-ΔKR (lane 4), or GST-ΔBromo (lane 5). (C) As in panel B, EMSA was performed with either GST alone (lanes 1 to 3) or WT GST-hbrm (lanes 4 to 15) with a 32-mer double-stranded oligonucleotide containing either 0, 10, or 24 A · T base pairs. When indicated, 300 ng of double-stranded poly(dG-dC) or poly(dA-dT) or 2 μM distamycin A was added to the reaction mixtures. (D) EMSA with either GST alone (lanes 1 and 2), WT GST-hbrm, or GST-hbrm DGD point mutant, using 32-mer double-stranded oligonucleotides containing either 0, 10, or 24 A · T base pairs as indicated.

FIG. 6

Deletion of the KR region of hbrm results in decreased affinity for nuclear structures. DT-derived cell lines expressing either WT hbrm, ΔCter(1337), ΔE7, ΔKR, or ΔBromo were fractionated by two different methods. In method 1 (A, B, D, F, H, J, and L), cells were lysed in a buffer containing 0.3% Nonidet P-40 and separated into supernatant (Sol fraction) and pellet. The pellet was treated with micrococcal nuclease and again centrifuged to separate supernatant (S1 fraction) and pellet. This pellet was further extracted with EDTA and centrifuged to yield supernatant (S2 fraction) and a pellet. This pellet was finally solubilized in 8 M urea (Pel fraction). One-tenth of each fraction was then extracted with phenol-chloroform and analyzed on a 1% agarose gel (A) or resolved directly by SDS-PAGE. For the latter, proteins were visualized by Western blotting with either anti-BRG-1 (B) or anti-hbrm (D, F, H, J, and L) antibodies. In method 2 (C, E, G, I, K, and M), nuclear matrix was prepared by the high-salt method as described in Materials and Methods. Cells were sequentially extracted with 0.5% Triton X-100 (Sol fraction), DNase I and 0.25 M (NH₄)₂SO₄ (CHR fraction), and 2 M NaCl, and the remaining pellet was solubilized in 8 M urea (Pel fraction). As in method 1, 1/10 of each fraction was subjected to SDS-PAGE and immunoblotted with anti-BRG-1 (C) or anti-hbrm (E, G, I, K, and M) antibodies. EtBr, ethidium bromide.