Activation of ATP-Binding Cassette Transporter A1 Transcription by Chromatin Remodeling Complex

From the Cardiovascular Research Institute (J.H., M.V., P.E.F., C.J.F.) and Departments of Medicine (P.E.F) and Physiology (C.J.F.), University of California, San Francisco.

Correspondence to Jarkko Huuskonen, University of California San Francisco, Cardiovascular Research Institute, Box 0130, San Francisco, CA 94143. E-mail jhuu6676@itsa.ucsf.edu

    Abstract

Objective— Liver X receptor (LXR) regulates the transcription of ATP-binding cassette transporter A1 (ABCA1) by binding to the DR-4 promoter element as a heterodimer with retinoid X receptor (RXR). The role of chromatin remodeling complex in LXR or ABCA1 activation has not been established previously. In this study, we investigated the activation of ABCA1 by brahma-related gene 1 (BRG-1) and brahma, members of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex.

Methods and Results— Overexpression of wild-type BRG-1 in SW-13 cells, but not a catalytically inactive mutant, increased ABCA1 mRNA levels determined by RT-PCR. These effects were enhanced by LXR and RXR agonists. In 293T (epithelial kidney cell line) and Hep3B (hepatocyte cell line) cells, small interfering RNA against BRG-1/brm also affected ABCA1 mRNA levels. Synergistic activation of ABCA1 was obtained after coexpressing BRG-1 and SRC-1, a coactivator of LXR. Luciferase assays showed that this activation of ABCA1 was dependent on the promoter DR-4 element. Coimmunoprecipitation and chromatin immunoprecipitation studies indicated that the mechanism of BRG-1–mediated activation of ABCA1 involved interaction of LXR/RXR with BRG-1 and binding of this complex to ABCA1 promoter.

Conclusions— Catalytic subunits of SWI/SNF chromatin remodeling complex, BRG-1 and brahma, play significant roles in enhancing LXR/RXR–mediated transcription of ABCA1 via the promoter DR-4 element.

Catalytic subunits of SWI/SNF chromatin remodeling complex, BRG-1 and brahma, enhanced ABCA1 transcription via the promoter DR-4 element. Physical interaction of LXR/RXR and BRG-1 and recruitment of BRG-1 to ABCA1 promoter was demonstrated. These results indicate that chromatin remodeling regulates ABCA1 transcription.

Key Words: ABCA1 ? HDL metabolism ? LXR ? chromatin remodeling ? BRG-1

    Introduction 

Eucaryotic DNA is assembled into nucleosomes. Their compact structure normally has a repressive effect on gene transcription. Two main classes of complexes exist that disrupt this chromatin structure and make it accessible for transcription factors and coregulators. One class covalently modifies histones and coregulators by changing the acetylation, phosphorylation, methylation, and ubiquitination patterns.1 The other class of chromatin modifiers uses ATP hydrolysis to disrupt histone–DNA interactions and change chromatin structure.2,3 These ATP-dependent chromatin remodeling complexes are divided into 4 main groups based on the relative similarity of the central ATPase subunit: the SWI/SNF (mating type switching/sucrose nonfermenting) family, the ISWI family, the CHD/Mi-2 family, and the Ino80 family.2,3

The best characterized complex of the chromatin remodeling complexes is human SWI/SNF. This complex always contains either BRG-1 (brahma-related gene 1; hSNF2?) or brm (brahma; hSNF2) as the catalytic subunit, together with 10 associated factors (BAFs).4 BRG-1 and brm share 70% sequence identity and enhance the transcription of genes regulated by several transcription factors, including nuclear hormone receptors (androgen receptor [AR], estrogen receptor [ER], glucocorticoid receptor [GR], progesterone receptor [PR], and retinoic acid receptor ), c-Myc, erythroid Kruppel-like factor (EKLF), CCAAT enhancer-binding protein beta (C/EBP?), and aryl hydrocarbon receptor/AHR nuclear translocator (AHR/ARNT).5–9 Recruitment of chromatin remodeling complex to target promoters is achieved either by direct interaction of BRG-1/brm with transcription factors or via bridging molecules such as p300/CREB-binding protein (CBP) and BAFs, which bind to nuclear receptors.4,10,11 BRG-1 and other subunits of mammalian SWI/SNF complexes also associate with retinoblastoma proteins and histone deacetylases exerting transcriptional repression.4

ATP-binding cassette transporter A1 (ABCA1) is a key regulator of high-density lipoprotein (HDL) metabolism.12 This membrane transporter facilitates the transfer of phospholipids to apolipoprotein A-I, the major protein constituent of HDL, thus initiating the formation of HDL. Several studies using either overexpression or inactivation of ABCA1 in mice have demonstrated the atheroprotective nature of this transporter.12 The transcriptional regulation of ABCA1 is complex and includes the use of alternative transcription start sites and competitive binding of several nuclear receptor dimers to the same DR-4 element in ABCA1 promoter.13–18 Liver X receptor (LXR) is a nuclear receptor that binds to the DR-4 element of target promoters as a heterodimer with retinoid X receptor (RXR).19 LXR/RXR heterodimers are activated by oxysterols (LXR ligands) and retinoic acid (RXR ligand) and can enhance the transcription of several important genes involved in lipid metabolism and inflammation.19 ABCA1 is activated by LXR and RXR agonists in vitro and in vivo.16,20 In mice, these agonists increase HDL levels and inhibit atherogenesis.21 In this study, we demonstrated that SWI/SNF chromatin remodeling complex interacts directly with LXR/RXR heterodimer complex and greatly enhances the transcription of ABCA1.

    Methods

A detailed Materials and Methods section is available online at http://atvb.ahajournals.org.

Transfections and Luciferase Assay

Transient transfections into SW-13 cells were performed using Fugene 6 (Roche) according to manufacturer instructions. One day after transfection, the medium was changed to DMEM with albumin and antibiotics, together with the other additions as shown for individual experiments. The final concentration for 22(R)-OH and 9-cis retinoic acid (CRA) was 10 μmol/L. After 24 hours, cells were harvested for experiments. Luciferase assays were performed using a dual-assay system.

RNA Interference in 293T and Hep3B Cells

Hep3B (hepatocyte cell line) and 293T (epithelial kidney cell line) cells were seeded on 24-well plates (1x105 for Hep3B and 2x105 cells for 293T) 1 day before transfection. The small interfering RNA (siRNA; synthesized by Dharmacon) targeting human BRG-1 and brm (sense sequence r(GCUGGAGAAGCAGCAGAAG)d(TT))22 was transfected into cells using Dharmafect 1 (Dharmacon) according to manufacturer instructions. Twenty-four hours after transfection, the media was changed as described above. RNA was isolated 48 hours after transfection and sodium dodecyl sulfate samples were collected at the 72-hour time point.

Reverse Transcription–Polymerase Chain Reaction

RT-PCR was performed as described previously.18

In Vivo Dithiobis(succinimidylpropionate) (DSP) Cross-Linking

Coimmunoprecipitation of LXR/RXR and BRG-1 from cross-linked 293 cells stably overexpressing FLAG-tagged LXR was performed as described previously.23,24

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays from 293 cells stably overexpressing FLAG-tagged LXR, 293T cells, and Hep3B cells were performed as described previously.25 Two improvements to the protocol were made. Cells were washed twice with PBS and first cross-linked 20 minutes at room temperature with 1.6 mmol/L DSP (see above) before formaldehyde cross-linking. This enhanced the binding of BRG-1 to the LXR/RXR complex. This is in agreement with Kurdistani et al, who found that histone deacetylase Rpd3 binding to the inositol-1-phosphate synthase (INO1) promoter was enhanced by previous cross-linking with several protein–protein cross-linkers.26 Thus, this double cross-linking method is especially useful in studying components of transcription factor complexes that do not directly interact with DNA. After lysing the cells, the nuclei were digested with 2 U of micrococcal nuclease for 10 minutes at +37°C in the same lysis buffer supplemented with 1.3 mmol/L CaCl2. The reaction was stopped by addition of 0.3 mmol/L EGTA. The micrococcal nuclease treatment of the nuclei before sonication made the chromatin more accessible and produced smaller, more uniform chromatin fragments, reducing the background of the ChIP assay. PCR was performed with Taq polymerase (Qiagen).

    Results

BRG-1 Upregulates ABCA1 mRNA Levels

To test whether human SWI/SNF chromatin remodeling complex plays a role in the transcriptional activation of ABCA1, we used adenocarcinoma cell line SW-13, which lacks the ATP-hydrolyzing subunits BRG-1 and brm of the complex.27 Western blotting of the nuclear extracts shows that these cells expressed nuclear receptors LXR and RXR, as well as coactivators SRC-1 and p300 and corepressors SMRT and nuclear receptor corepressor (NCoR) (please see online supplement). Each of these coregulators had been shown to influence ABCA1 transcription in previous studies.25,28,29 The LXR and RXR ligands, 22(R)-OH and 9-CRA, respectively, did not have a major influence on the amount of LXR, RXR, or coregulators in SW-13 cells. These results indicate that SW-13 cells contain the normal coregulators of ABCA1.

When SW-13 cells were incubated with 22(R)-OH or 9-CRA, ABCA1 mRNA was increased 8-fold (Figure 1). Simultaneous addition of these ligands had a synergistic effect on ABCA1 levels. In the absence of LXR or RXR ligands, overexpression of wild-type BRG-1, but not the mutant BRG-1 deficient in ATP-hydrolyzing function, upregulated ABCA1 expression levels 50-fold. The synergistic effect of LXR and RXR ligands was retained. The expression levels of the BRG-1 wild-type and mutant proteins were equal on transfection (Figure 1, inset). The basal- and 22(R)-OH–induced ABCA1 levels were increased up to 100-fold, with gradually increasing BRG-1 levels (please see online supplement). These data demonstrate that the BRG-1 subunit of SWI/SNF complex plays a major role in upregulating ABCA1 transcription.

   Figure 1. Upregulation of ABCA1 mRNA levels by BRG-1. SW-13 cells were transfected with either BRG-1 wild-type (wt) or K798R mutant BRG-1 (mut) and incubated after transfection either under noninducing condition (co) or induced with LXR ligand 22(R)-OH (OH; 10 μmol/L), RXR ligand 9-CRA (CRA; 10 μmol/L) either alone or in combination (comb; 10 μmol/L each) for 24 hours. Nontransfected cells (control) were used as a reference. RNA from these cells was isolated, reverse transcribed, and real-time PCR was performed using oligonucleotides specific for ABCA1. mRNA levels (+SD) relative to noninduced control cells is presented from a representative experiment performed in triplicate. Inset shows the expression levels of wild-type and mutant BRG-1 detected by Western blotting from cell lysates.

In the human SWI/SNF chromatin remodeling complex, the ATP-hydrolyzing subunit is either BRG-1 or brm. When the expression plasmid for brm was transfected into SW-13 cells, similar results of ABCA1 mRNA levels were observed compared with BRG-1 overexpression (data not shown). This indicates that either of the SWI/SNF subunits (brm or BRG-1) activates LXR/RXR-mediated transcription of ABCA1.

Next, we tested whether other LXR-regulated genes were also affected by BRG-1 expression. In BRG-1–overexpressing SW-13 cells, phospholipid transfer protein (PLTP) was modestly upregulated (3.4±1.3-fold induction; P<0.01; n=6), whereas sterol regulatory element-binding protein 1c (SREBP1c) was downregulated (mRNA levels 0.4±0.2 compared with control; P<0.001; n=9). In either case, the expression levels of these genes were not affected by LXR ligand 22(R)-OH in BRG-1–overexpressing cells (data not shown). These results indicate that SWI/SNF chromatin remodeling complex is able to influence the expression of several LXR-regulated genes.

ABCA1 Is Regulated by BRG-1 in Several Cell Types

The effect of BRG-1 on ABCA1 expression was demonstrated in 2 other cell lines: 293T and Hep3B. When siRNA directed toward BRG-1 and brm was transfected into 293T cells, mRNA levels of these genes were reduced by 60% to 70% (Figure 2A). There was a comparable reduction of BRG-1 and brm protein levels in these cells (Figure 2A, inset). The reduction of BRG-1/brm affected ABCA1 levels; in noninducing cells, the basal level of ABCA1 was increased 3-fold (Figure 2B, left). Most importantly, the ligand-mediated induction of ABCA1 in 293T cells was attenuated 45% by reducing BRG-1/brm levels (Figure 2B, right).

   Figure 2. Effect of BRG-1/brm siRNA on ABCA1 levels. 293T cells were transfected with siRNA (+siRNA) against BRG-1/brm and incubated after transfection either under noninducing condition (co) or induced with LXR ligand (OH). Mock-transfected cells (–siRNA) were used as a reference. A, Average mRNA levels (+SD) of BRG-1 (black bars) and brm (white bars) from 3 independent experiments is shown relative to nontransfected cells under noninducing conditions. Numbers refer to BRG-1 levels. Inset shows the protein levels of BRG-1 and brm from a representative experiment. **P<0.01 and *** P<0.001 of –siRNA vs +siRNA. B, Average ABCA1 mRNA levels (+SD) in 293T cells from 3 independent experiments. Left, Increase in basal ABCA1 levels with BRG-1/brm siRNA under noninducing conditions (**P<0.01 –siRNA vs +siRNA). Right, Decrease in ABCA1 levels on LXR ligand induction with BRG-1/brm siRNA (***P<0.001 –siRNA vs +siRNA).

Similar effects on the basal ABCA1 levels were observed in Hep3B cells. Increased ABCA1 levels (mRNA with siRNA 1.7±0.2 versus no siRNA; P<0.05; n=3) were observed when BRG-1 and brm mRNA levels were reduced by 80% and 70% (P<0.01 versus no siRNA for BRG-1 and brm; n=3), respectively. Similarly to published data on another hepatocyte cell line (HepG2), ABCA1 levels were not induced by 22(R)-OH,30 and therefore, the effect of siRNA on hydroxycholesterol-mediated induction could not be measured in Hep3B cells. Together, the results from siRNA studies demonstrate that the effect of BRG-1 on ABCA1 mRNA levels is not restricted to SW13 cells but can be observed in other cell types as well.

BRG-1 Effect on ABCA1 Transcription Is Dependent on Promoter DR-4 Element

To localize the promoter element responsible for the BRG-1–mediated upregulation of ABCA1, we used ABCA1 luciferase constructs. In agreement with the mRNA data, ABCA1 promoter activity was increased by LXR and RXR ligands and by overexpression of wild-type BRG-1 (Figure 3). Surprisingly, and in contrast with endogenous mRNA, mutant BRG-1 was also capable of inducing ABCA1 transcription from a plasmid template. The major effect of LXR and RXR ligands in ABCA1 promoter has been attributed to the DR-4 element.16,18,20,25 When this element was mutated from the full-length promoter construct, the ligand- and BRG-1–dependent effects were significantly reduced (Figure 3), demonstrating the importance of this element for ABCA1 transcription.

   Figure 3. ABCA1 promoter activity is increased by wild-type and mutant BRG-1 in DR-4–dependent manner. SW-13 cells were transfected either with 500 ng GFP (control), BRG-1 wild-type, or mutant expression plasmids, together with 500 ng of ABCA1 promoter construct and 100 ng of pRL-TK (renilla luciferase cDNA under thymidine kinase promotor) internal control and incubated as described in Figure 1 legend. Cells were lysed in passive lysis buffer, and luciferase activities were measured using dual-luciferase assay system. The luciferase activities of wild-type (wt) ABCA1 promoter (black bars) and full-length ABCA1 promoter in which the DR-4 element was mutated (ABCA1-DR4; white bars) were calculated by normalizing the values to renilla luciferase values. The results are relative (+SD) to cells transfected with GFP under basal conditions. A representative experiment performed in triplicate is shown. The values refer to wild-type ABCA1 promoter construct. OH indicates 22(R)-OH; CRA, 9-CRA; comb, combination of 22(R)-OH and 9-CRA.

Steroid Receptor Coactivator 1 and BRG-1 Synergistically Activate ABCA1 Transcription

We reported previously that coactivators steroid receptor coactivator 1 (SRC-1) and p300 induced ABCA1 transcription in 293T cells.25 When SW-13 cells were transfected with SRC-1, basal ABCA1 levels were increased 4-fold (4.1±1.8; P<0.05; n=5). Similar activation was obtained with p300 overexpression (3.7±1.4; P<0.05; n=5). Overexpression of these proteins seemed to have a more dramatic effect on 9-CRA–mediated induction than on 22(R)-OH induction because an additional 3- to 5-fold increase of ABCA1 mRNA was observed (P<0.05; n=4 for CRA versus SRC-1+CRA and CRA versus p300+CRA; Figure 4; and data not shown). We also tested whether SRC-1 could further induce BRG-1–mediated activation of ABCA1. SRC-1+BRG-1 coexpression resulted in an additional 2- to 3-fold increase in ABCA1 basal levels compared with BRG-1 transfection alone (Figure 4). This effect was retained in cells induced with LXR or RXR ligands. These data indicate that SRC-1 and BRG-1 synergistically activate ABCA1 transcription.

   Figure 4. SRC-1 and BRG-1 synergistically upregulate ABCA1 expression. SW-13 cells were transfected with expression plasmid encoding SRC-1 (8 μg per transfection) or BRG-1 (4 μg per transfection) and incubated with various agents as explained in Figure 1 legend. mRNA levels of ABCA1 were determined using real-time RT-PCR and are shown relative to the nontransfected (control) cells under basal condition. A representative experiment performed in triplicate is shown.

Interaction of LXR and BRG-1 in Intact Cells

To determine the mechanism by which chromatin remodeling complex affects LXR-mediated regulation of ABCA1, we studied the interaction of LXR and RXR with BRG-1. Immunoprecipitation from cross-linked LXR–overexpressing cells with specific antibodies against FLAG (precipitating epitope-tagged LXR) and RXR coprecipitated BRG-1 protein (Figure 5A). This interaction was present in noninduced cells and in cells incubated with 22(R)-OH but was not observed when the immunoprecipitation was performed using nonspecific rabbit IgG. These results indicate specific interaction between LXR-BRG-1 and RXR-BRG-1. In separate experiments, we were also able to show the interaction between LXR and RXR (data not shown).

   Figure 5. In vivo interaction of LXR/RXR and BRG-1. A, Coimmunoprecipitation of LXR/RXR and BRG-1. 293 cells stably overexpressing LXR under basal (control) or inducing (2-hour incubation with 10 μmol/L 22(R)-OH) were cross-linked with DSP. Coimmunoprecipitation was performed as described in Methods section using specific antibodies against FLAG (precipitates LXR), RXR, BRG-1, or with nonspecific rabbit IgG. Presence of BRG-1 in the coimmunoprecipitations was assayed by Western blotting with anti–BRG-1 antibody. B, Chromatin immunoprecipitation. 293-LXR, 293T, and Hep3B cells were grown under basal conditions or induced with 22(R)-OH for 2 hours, fixed with DSP and formaldehyde, and ChIP assay using anti-FLAG (for tagged LXR in 293–LXR cells), anti-LXR (293T and Hep3B cells), anti-RXR, and anti-BRG-1 antibodies was performed as described in Methods section. The promoter region of ABCA1 surrounding the DR-4 element was amplified by PCR using gene-specific primers. H and G refer to negative (water as template) and positive (100 ng of genomic DNA) PCR controls, respectively. B refers to negative control of the ChIP assay (buffer precipitated with nonspecific rabbit IgG).

We also performed ChIP studies in several cell types. Specific binding of LXR, RXR, and BRG-1 to the DR-4 element of the ABCA1 promoter was demonstrated in 293 cells stably overexpressing LXR, in 293T cells and in Hep3B cells (Figure 5B). In agreement with coimmunoprecipitation data, the binding of BRG-1 to ABCA1 promoter was present in the absence and presence of LXR agonist. Together, these results demonstrate the recruitment of BRG-1 to the DR-4 element of the ABCA1 promoter and physical interaction of LXR/RXR and BRG-1.

    Discussion

Regulation of ABCA1 expression is determined by multiple mechanisms. Protein and mRNA stability play a role together with the use of multiple transcription start sites and competitive binding of different nuclear receptor heterodimers (LXR/RXR, RAR/RXR, TR/RXR) to the same promoter DR-4 element.13–18,31 Certain coregulators, such as SRC-1/p300, NCoR/SMRT, RIP140, MBF-1, ASC-2, SHP, and PGC-1 have been shown to affect LXR and, in some cases, also ABCA1 activity.25,28,29,32–36 However, the role of chromatin remodeling complex on the activation of LXR or ABCA1 has not been determined.

SWI/SNF chromatin remodeling complex affects the transcription of 5% of genes in yeast.3 The catalytic subunits BRG-1 and brm have overlapping but distinct functions because BRG-1 knockout mice are lethal, whereas brm-deficient mice are overweight but otherwise apparently normal.37 BRG-1 and brm activate nuclear hormone receptors and hypoxia-inducible factor 1.1,9,22 On the contrary, BRG-1 preferentially binds and activates zinc finger proteins (including RXR and RAR), whereas brm activates factors with 2 ankyrin repeat proteins.37 In the current study, overexpression of either wild-type BRG-1 or brm activated LXR/RXR-mediated transcription of ABCA1 in SW-13 cells that normally lack these proteins (Figure 1). This indicates that human SWI/SNF subunits can compensate for each other in the activation of LXR/RXR. We also demonstrated that not only ABCA1, but 2 other LXR-regulated genes (SREBP1c and PLTP), were affected by BRG-1. mRNA levels of ABCA1 and PLTP were upregulated in SW-13 cells, but SREBP1c was downregulated by BRG-1 overexpression. More research is needed to determine the mechanism behind the differential effect on these LXR-regulated genes.

The regulation of ABCA1 by BRG-1 was not restricted to SW-13 cells but was also observed in kidney epithelial and hepatocyte cell lines (Figure 2). Most importantly, the upregulation of ABCA1 mRNA levels by LXR ligand was attenuated by 45% when BRG-1 and brm levels were decreased using siRNA (Figure 2B, right). This is in agreement with our findings in which ABCA1 levels were greatly upregulated by LXR and RXR ligands in BRG-1 overexpressing SW-13 cells (Figure 1). In contrast, regulation of basal ABCA1 expression by BRG-1 seems to differ between cell types because overexpressing BRG-1/brm in SW-13 cells leads to increased ABCA1 expression, whereas a similar effect was seen in 293T and Hep3B cells when the level of both proteins was decreased by siRNA (Figures 1 and 2). BRG-1/brm may affect the basal activity of ABCA1 differently in these cells via other transcription factors than LXR/RXR.

Activation of ABCA1 by oxysterols and retinoic acid was preserved in SW13 cells even in the absence of SWI/SNF chromatin remodeling activity (Figures 1, 3, and 4), a result observed previously in the response of the probasin gene to AR receptor ligand DHT, and the response of GR to its ligand DHT.9,27 These data indicate that for some nuclear receptors, ligand-mediated transcriptional activation can occur even in the absence of chromatin remodeling activity. However, the activation is greatly enhanced by the presence of catalytically active subunits of the SWI/SNF complex.

Measurement of endogenous mRNA levels reflect activity on native chromatin contex. This conformational complexity is lacking in plasmid templates, which are, however, widely used and offer useful tools in deciphering the elements responsible for transcriptional effects. Luciferase assays were used to localize the BRG-1–responsive element in the ABCA1 promoter. When the DR-4 element in the promoter was mutated, the BRG-1 effect was abolished, indicating the essential role of this element for activation (Figure 3). An unexpected finding was that mutant BRG-1 induced ABCA1 promoter activity almost to the same extent as the wild-type BRG-1. This had been reported earlier for BRG-1–upregulated GR-responsive mouse mammary tumor virus (MMTV) promoter, as well as for CYP1A1 promoter activated by AHR/ARNT.5,8,27 The explanation probably lies in the differential structure of the "open" template (plasmid) versus "compact" template (native chromatin). In the case of the plasmid template, the ATPase-deficient form of BRG-1 can enhance the transcription by additional mechanisms, such as facilitating interactions between nuclear receptors and other coactivators, or stabilizing interaction with RNA polymerase II.5,27 The difference between mutant BRG-1–mediated activation of ABCA1 on 2 different templates (no activation on "compact" native chromatin template versus activation on "open" plasmid template) also implies that measuring luciferase activity alone may not be sufficient to draw conclusions about transcriptional mechanisms involving chromatin remodeling.

The mechanism by which SWI/SNF complex enhanced the LXR/RXR-mediated transcription of ABCA1 involves the DR-4 element of the promoter, as well as interaction of BRG-1 with LXR/RXR complex. This was demonstrated by deleting the DR-4 element on of the ABCA1 promoter construct (Figure 3), as well as coimmunoprecipitation and ChIP data (Figure 5). The interaction between other nuclear receptors and BRG-1 has been reported previously to be either direct (interaction of BRG-1 with RAR and RXR)37 or indirect, involving coactivators SRC-1/p300 (AR and TR)11 or BAFs (GR and ER).1 Because our coimmunoprecipitations were performed using cross-linked cells, we cannot distinguish between direct interaction of LXR/RXR with BRG-1 versus indirect recruitment of BRG-1 by SRC-1/p300/BAFs. However, we provide further evidence that LXR/RXR, SRC-1, and BRG-1 are all part of the same transcriptional complex for ABCA1. SRC-1 and BRG-1 synergistically activated ABCA1 transcription (Figure 4). This stimulatory effect of SRC-1 was also localized to the ABCA1 DR-4 element (data not shown). The synergy between BRG-1 and SRC-1 or CBP (which has acetyltransferase activity like p300) was reported previously for ER-responsive promoters6 and for CYP1A1 activation by AHR/ARNT heterodimer.8

A unique feature of LXR compared with steroid nuclear receptors (such as GR and ER)6,27 is that the interaction of BRG-1 with LXR complex was ligand independent (Figure 5). Two explanations for this difference are plausible. Unlike steroid receptors, which are transported to nucleus and recruited to promoter elements as homodimers on ligand activation, RXR heterodimers are mainly found in the nucleus and permanently occupy promoters. This indicates a basic difference in the promoter occupancy between steroid receptors and RXR heterodimers. Kadam et al also reported direct, ligand-independent interaction between RXR and BRG-1,37 a result consistent with our findings. Second, whereas steroid receptor ligands (such as ERs and GRs) can easily be depleted from cell cultures (because no endogenous ligands are produced by most cells), LXR ligands are produced endogenously by several cell types. Thus, it is possible that small amounts of oxysterols produced in the cell types used provide endogenous signals for BRG-1 binding to LXR/RXR complex.

In summary, our results show that SWI/SNF chromatin remodeling complex plays a major role in the activation of ABCA1 via LXR/RXR. This activation is mediated by the promoter DR-4 element and involves direct association of LXR/RXR complex and BRG-1. These results are compatible with a model in which the activation of ABCA1 transcription involves LXR/RXR heterodimer binding to DR-4 element and recruitment of coactivators SRC-1/p300 and SWI/SNF chromatin remodeling complex (Figure 6).

   Figure 6. Model of transcriptional activation of ABCA1. In basal SW-13 cells, RXR/LXR heterodimer is bound to the ABCA1 DR-4 element with no coactivators. No transcription/basal activity is observed. On either LXR or RXR ligand activation, coactivators SRC-1 and p300 are able to bind to LXR/RXR, and modest RNA polymerase II (RNA pol II)–mediated activation is observed. When SWI/SNF chromatin complex subunits BRG-1 or brm are introduced into the cells, major rearrangement of the nucleosomal chromatin facilitates recruitment of BAFs. Maximal trascriptional activity is observed. Gray coloring indicates factors that are associating/playing a role in each activation step.

    Acknowledgments

This work was supported by National Institutes of Health Grants HL57976 and HL67294 (P.F., C.F.).

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