Liver X Receptors Interact with Corepressors to Regulate Gene Expression

Xiao Hu, Suzhen Li, Jun Wu, Chunsheng Xia and Deepak S. Lala

Department of Biotechnology, Pharmacia Corp., St. Louis, Missouri 63017

Address all correspondence and requests for reprints to: Deepak S. Lala, Ph.D., or Xiao Hu, Ph.D., Mail Zone AA3G, 700 Chesterfield Parkway North, Chesterfield, Missouri 63017. E-mail: Deepak.S.Lala{at}pharmacia.com or Xiao.Hu{at}pharmacia.com.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate gene expression in response to oxysterols and play a critical role in cholesterol homeostasis by regulating genes that are involved in cholesterol transport, catabolism, and triglyceride synthesis. Oxysterols and synthetic agonists bind LXRs and activate transcription by recruiting coactivator proteins. The role of LXRs in regulating target gene expression in the absence of ligand is unknown. Here we show that LXRs interact with corepressors, N-CoR (nuclear receptor corepressor) and SMRT (silent mediator of retinoic acid receptor and thyroid receptor), which are released upon binding agonists. The LXR-corepressor interaction is isoform selective, wherein LXR{alpha} has a very strong interaction with corepressors and LXRß only shows weak interaction. LXRs also exhibit a preference for interacting with N-CoR vs. SMRT. Similar to other nuclear receptors, mutations in the LXR helix 3 and 4 region abolish corepressor interaction. Using a transient transfection assay, we demonstrate that LXR represses transcription that can be further increased by cotransfecting N-CoR into cells. Chromatin immunoprecipitation experiments further indicated that N-CoR is recruited onto endogenous LXR target genes, and addition of LXR agonists releases N-CoR from their promoters. Collectively, these results suggest that corepressors play an important role in regulating LXR target gene expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS ARE a class of DNA binding transcription factors that regulate gene expression in response to ligand and play very important roles in many biological and pathological processes. Consistent with this, these proteins serve as important drug targets, and indeed several members of this class are targets for existing drugs. Agonist-occupied nuclear receptors, when bound to their cognate DNA response elements, generally activate transcription by recruiting coactivators (1). Antagonist occupied nuclear receptors, on the other hand, either block transcriptional activation or actively repress transcription (1, 2). A subset of nuclear receptors, including thyroid receptor (TR) and retinoic acid receptor (RAR), actively repress transcription in the unliganded state. This is most likely due to their ability to interact with corepressor proteins, such as N-CoR and silent mediator of retinoic acid receptor and thyroid receptor (SMRT) in the absence of ligand (3, 4). These corepressors are large proteins that share extensive homology in the repression domain at the N-terminal region and in the receptor interaction domain at the C-terminal region. Both N-CoR and SMRT repress transcription by recruiting histone deacetylases through their respective repression domains (5, 6, 7, 8, 9). The receptor interaction domains of N-CoR and SMRT contain small peptide motifs (CoRNR boxes) that are both necessary and sufficient for interactions with TR, RAR, and, under some conditions antagonist bound estrogen receptor (10, 11, 12, 13, 14). The specificity of nuclear receptor-corepressor interaction is determined by the individual nuclear receptor interacting with specific CoRNR boxes within a preferred corepressor (15, 16). Initial mutagenesis studies suggested that corepressors and coactivators interact with the nuclear receptor ligand binding domain (LBD) via similar yet distinct amino acid sequences (10, 11, 12). Additionally, recent x-ray crystallographic studies have shown that corepressors bind to the coactivator binding groove on the LBD of the receptor (17).

LXR{alpha} (NR1H3) and LXRß (NR1H2) are members of the nuclear receptor superfamily that heterodimerize with the retinoid X receptor (RXR) and are activated via modified cholesterol molecules that function as endogenous ligands (18). The identification of synthetic LXR specific ligands has greatly facilitated the discovery of LXR regulated genes (19). Both LXR{alpha} and LXRß have been shown to regulate several important genes in reverse cholesterol transport, including the ATP-dependent cholesterol transporter (ABCA1), cholesteryl ester transfer protein, apolipoprotein E, and lipoprotein lipase in vitro and in vivo (20, 21, 22, 23, 24). Consistent with their ability to activate reverse cholesterol transport, full agonists of LXR have also been reported to increase HDL in mice. LXR agonists have the potential to eliminate the accumulation of lipid by direct activation of key genes in macrophages to eventually reduce atherosclerotic lesions (25, 26). In addition to the beneficial effects on cholesterol efflux, LXR ligands that function as full agonists also increase gene expression of sterol response element binding protein (SREBP1c), a key gene that activates transcription of major genes involved in triglyceride synthesis (e.g. fatty acid synthase) (19, 27). Consistent with this, administration of LXR full agonists causes a dramatic increase in triglyceride synthesis in rodents. All the above studies were done using either natural LXR ligands (oxysterols) or synthetic LXR agonists. The exact role of LXRs in the absence of ligands is still unknown. While knockout studies have indicated that the expression levels of several LXR target genes are reduced in liver, it is intriguing that at least some genes, for example, SREBP-1, appear to be increased in white adipose tissue of LXR{alpha}/ß double knockout mice (27). This suggests that removal of LXR can, at least under certain circumstances, lead to derepression of its target genes. A potential explanation for the tissue-selective responses might be that LXRs selectively interact with corepressors in different tissues perhaps as a consequence of varying levels of endogenous ligands in different tissues. Consistent with this hypothesis, SREBP-1 expression is higher in liver than in white adipose when wild-type mice were fed with chow diets (27). Finally, other RXR partners, such as RAR, TR, and peroxisome proliferator-activated receptor (PPAR){delta} interact with corepressors in the absence of agonists. Together, these data prompted us to explore the role of corepressors in LXR-mediated gene regulation.

In this report, we show that LXRs are capable of interacting with the corepressors N-CoR and SMRT, although the interaction with SMRT is much weaker. The corepressor interaction surface on LXR is similar to that identified in TR and RAR. In the absence of ligand, LXRs repress transcription of reporter genes, and this repression can be further increased by cotransfecting N-CoR into the cells. We also show by chromatin immunoprecipitation (ChIP) experiments, that N-CoR is associated with endogenous LXR target gene promoters in the absence of ligand, and addition of a LXR agonist releases N-CoR binding. These results suggest that corepressors play a key role in controlling LXR modulation of its target genes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In an attempt to understand the molecular mechanisms involved in LXR function, we used a mammalian two-hybrid assay to investigate if LXRs interact with corepressors in the absence of ligand. The receptor interaction domain of N-CoR was fused to a GAL4 DNA binding domain and full length LXRs were fused to a VP16 activation domain. As shown in Fig. 1AGo, both LXR{alpha} and LXRß interact with the C-terminal receptor interaction domain of N-CoR. The magnitude of interactions is comparable to that of RAR and N-CoR (Ref. 15 ; and data not shown). These interactions were observed in the absence of ligand and LXR agonists, either its endogenous ligand 22R-hydroxycholesterol or the synthetic ligand T0901317, almost completely released the corepressor. It is also worth noting that the interaction of N-CoR with LXR{alpha} is much stronger than that with LXRß, suggesting a difference in the ability of the two isoforms to recruit corepressors.



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Figure 1. LXRs Interact with Corepressor N-CoR

A, The receptor interaction domain (ID) of N-CoR interacts with LXRs in mammalian two-hybrid assay. Ligand used, 22R-OHC (22R-hydroxycholesterol, 100 µM), T0901317 (10 µM). B, Each CoRNR box peptide interacts with LXRs. Top, interaction with CoRNR1. Bottom, interaction with CoRNR2. RAR{alpha} and PPAR{delta} were included for comparison. C, LXR{alpha} interacts directly with CoRNR peptide in vitro in ALPHA screen. D, LXR{alpha} (Top) and LXRß (Bottom) interact differentially with N-CoR and SMRT.

 
The interaction domains present within corepressors contain two CoRNR box motifs and each CoRNR motif is necessary and sufficient for interaction with unliganded receptors (10, 11, 12). Different receptors exhibit distinct preferences for these two CoRNR motifs, with RXR and PPAR showing preference toward CoRNR2, and RAR toward CoRNR1 (15). We therefore tested whether LXRs exhibit similar preferences with respect to these interaction motifs. As shown in Fig. 1BGo (top and bottom), in contrast to RAR{alpha}, which strongly interacts with CoRNR1, and PPAR{delta}, which only interacts with CoRNR2, LXR{alpha} and LXRß interact with both CoRNR motif peptides. However, the interactions with the two CoRNR motif peptides are not equivalent. Whereas the magnitude of the LXR CoRNR1 interaction is comparable to that of RAR{alpha}, the interaction with CoRNR2 is much stronger. Under similar conditions, LXR{alpha} showed more than a 1000-fold interaction with CoRNR2, whereas PPAR{delta} only showed about a 40-fold interaction. Moreover, and consistent with Fig. 1AGo, LXR{alpha} displayed a tighter association with both CoRNR motif peptides than LXRß, further confirming the observation that the two isoforms exhibit unique preferences for their interactions with corepressor motifs.

Having established an interaction between LXR and corepressors in cells, we next used biochemical peptide recruitment assays to demonstrate a direct interaction between LXR and corepressor peptides. A biotinylated CoRNR1 peptide was incubated with bacterially produced glutathione-S-transferase (GST)-tagged LXR{alpha} protein in the presence or absence of T0901317. The interaction was detected using a labeled GST antibody in an amplified luminescent proximity homogeneous assay (ALPHA). When LXR{alpha} protein binds the peptide, the luminescent dye on the antibody is in close proximity to the dye associated with the peptide (via biotin-streptavidin), and a signal is released. As shown in Fig. 1CGo, the signal derived from a control peptide is not altered by adding T0901317. In contrast, this signal is much higher (4–5x) when LXR{alpha} LBD is incubated with the CoRNR1 peptide, indicating a direct and specific interaction between CoRNR1 and LXR{alpha}. Addition of T0901317 reduces this signal to background level, indicating the interaction between CoRNR1 peptide and LXR{alpha} is disrupted by ligand. These biochemical experiments demonstrate a direct interaction of LXR{alpha} and corepressor proteins in the absence of ligand and a ligand-dependent release of corepressors from LXR.

Specific nuclear receptors also display selective preferences for the different corepressors. For example, TR prefers N-CoR, whereas RAR interacts better with SMRT. We therefore tested whether LXRs also exhibit any preferences in regard to corepressor interactions. As shown in Fig. 1DGo, SMRT, compared with N-CoR, showed a much weaker interaction with both LXR{alpha} (Top) and LXRß (Bottom). Under similar conditions, RAR{alpha} has been shown to interact better with SMRT (15, 28).

The above data suggest that LXRs can recruit corepressors in the absence of ligand and are highly selective in nature. Based on these observations, we speculated that LXRs would repress transcription when ligand is not available. To test this hypothesis, we used a construct containing GAL4 DBD fused with LXR LBD. When cotransfected with a reporter containing GAL4 binding sites, both LXRs repressed reporter gene expression in HEK293 cells (Fig. 2AGo). The fold repression mediated by LXR{alpha} is comparable to that of RAR{alpha}, a well-known receptor that also represses transcription in its unliganded state. Consistent with its weak interaction with corepressors, LXRß showed relatively weaker repression. It should be noted that lipoprotein-free serum was used in these experiments. When normal serum was used, little repression was detected presumably due to the presence of some LXR ligands in normal serum. To further demonstrate that corepressors play a role in the repression, we introduced exogenous corepressors into cells. As shown in Fig. 2BGo, cotransfection of N-CoR further increased the repression by LXR{alpha}.



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Figure 2. LXRs Repress Transcription

A, GAL4 fusions of LXR LBDs repress transcription on a GAL4 response element-luciferase reporter construct. B, N-CoR increases the repression by GAL4-LXR{alpha}.

 
It has been previously shown that the corepressor interaction surface on nuclear receptors consists of helices 3–5 within the LBD, a region that is also involved in interactions with coactivator proteins. Several critical surface residues within this region have been shown to be involved in both coactivator and corepressor interactions (10, 29). To test whether similar amino acids present within LXRs are also critical for their interactions with corepressors, we made mutations in two residues, Val 269 and Ile287 within the LXR{alpha} LBD. As shown in Fig. 3Go, mutations in either of these two residues dramatically decreased the interaction of LXR{alpha} with N-CoR. These mutations did not cause a decrease in expression levels or destruction of structural integrity as these mutations did not alter the ability of LXR{alpha} to interact with RXR (data not shown).



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Figure 3. The Corepressor Interaction Surface on LXR Is Similar to that of TR

Top, Sequence alignments of H3–5 region of TR and LXR{alpha}. The two mutations (V269R and I287R) are shown below the alignment. Bottom, Mutations in H3–5 abolish corepressor interaction.

 
These experiments establish that LXR LBDs interact with corepressors in mammalian two-hybrid assay and repress transcription when fused to a GAL4 DBD in the absence of ligand. To extend the relevance of these observations to a more physiological setting, we investigated whether corepressors modulate LXR function when LXR is bound to its response elements (LXREs). Three copies of LXREs from either the human ABCA1 or SREBP1c promoter were cloned upstream of a synthetic minimal promoter and a luciferase gene. When transfected into HEK293 cells, these elements showed higher levels of transcription activity as compared with vector in the absence of ligand. The activation function 1 (AF1) region of LXRs could possibly contribute this basal activity. It should be pointed out that HEK293 cells express LXR{alpha} and several coactivators and corepressors as detected by Taqman analysis (data not shown). Northern analysis also indicated that LXR{alpha} gene is expressed at high levels in both liver and kidney (30). The presence of functional LXR proteins in HEK293 cells is also demonstrated by the ability of T0901317 to activate these LXRE-containing reporters (Fig. 4AGo). These data suggest that endogenous LXR can activate the LXRE driven promoters in a ligand-dependent manner. When an expression construct of N-CoR was cotransfected along with these LXRE reporters, the reporter activities were significantly decreased (Fig. 4BGo). In contrast, cotransfection of N-CoR had no effect on the expression of the control reporter lacking LXREs. This effect of N-CoR on LXRE-containing reporters but not on a control reporter lacking an LXRE suggests that N-CoR is recruited to the LXRE, most likely via endogenous LXR. Consistent with the weak interaction of SMRT with LXR, cotransfection of SMRT had little effect on the LXRE driven promoter under these conditions.



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Figure 4. N-CoR Decreases Transcription of LXRE-Containing Reporters

A, T0901317 activates LXRE-containing reporters. B, Corepressor preferences of LXRs. N-CoR but not SMRT decreases basal activity of LXREs derived from ABCA1 and SREBP1c promoters. Three copies of LXREs from either ABCA1 or SREBP1c promoter were cloned upstream of a luciferase reporter. The transcriptional activity of each reporter was monitored under various cotransfection conditions. "Relative Luc Activity" here is relative to the basal activity of the vector.

 
Next, we investigated whether LXRs recruit corepressors on endogenous LXR target genes. It has been shown that LXRs regulate expression of ABCA1 and SREBP1c gene expression in human macrophage cell line THP1 and liver cell line HepG2 respectively (31, 32). To determine if these genes are regulated by LXR in HEK293 cells, we used real-time PCR (Taqman) to examine their transcript levels. As shown in Fig. 5AGo, when HEK293 cells are treated with T0101317, both ABCA1 and SREBP1c genes are induced, further indicating that LXR is functional in these cells and capable of activating its target genes.



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Figure 5. N-CoR Is Recruited to Endogenous ABCA1 and SREBP1c Genes in HEK293 Cells

A, LXR agonist induces ABCA1 and SREBP1c genes in HEK293 cells. Expression levels of ABCA1 and SREBP1c were examined by Taqman analysis. B, ChIP analysis of ABCA1 and SREBP1c genes. HEK293 cells were transfected with Flag-tagged N-CoR and treated with LXR specific agonist, T0901317, at 10 µM. The occupancy of ABCA1 and SREBP1c promoter was monitored by PCR using specific primers surrounding the LXRE in each gene. The numbers (relative to transcription start site) in the diagram indicate the position of primers.

 
Having established that LXR regulation of ABCA1 and SREBP1c is well preserved in HEK293 cells, we next used ChIP assay to determine if corepressors are recruited to these two genes by LXR. Due to the lack of reliable N-CoR antibodies, we first transfected HEK293 cells with Flag-tagged N-CoR and then used a Flag antibody to immunoprecipitate chromatin DNA. As shown in Fig. 5BGo, LXR{alpha} bound to the promoters of both ABCA1 and SREBP1c genes, and addition of LXR agonist T0901317 had no significant effect on the binding of LXR{alpha} to its target promoters. In contrast to LXR{alpha}, N-CoR, which was also bound to the promoters, was released from the promoters after addition of T0901317. Our results suggest that corepressors are recruited to target gene promoters by LXR in the absence of agonists. Upon addition of full agonists such as T0901317, the corepressors are released, leading to transcriptional activation of LXR target genes.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LXRs were initially classified as orphan receptors because their ligands had not been identified. The role of LXRs in lipid regulation was gradually established after knockout studies and the discovery of oxysterols as endogenous LXR agonists. Subsequent studies have also demonstrated a role for LXR in atherosclerosis because the absence of the receptors in macrophages leads an increase in foam cell formation (33). The LXR agonist T0901317 has also been shown to reduce atherosclerotic lesion size in genetic models of atherosclerosis (25, 26). Collectively, these data suggest that LXRs may serve as novel targets for the treatment of atherosclerosis and other lipid disorders.

LXRs belong to a subgroup of the nuclear receptors that function as heterodimers with RXR. Although many receptors in this class, such as TR and RAR have been shown to associate with corepressors in the absence of their cognate ligands, the role of LXR in the absence of its agonists is unknown. Here we report that LXRs, like TR and RAR interact with corepressors, especially N-CoR via their LBDs in the absence of ligand and are released upon addition of an agonist. The corepressors N-CoR and SMRT contain two CoRNR boxes (CoRNR1 and CoRNR2) that control the recruitment of nuclear receptors and these receptors exhibit distinct preferences for CoRNR1 vs. CoRNR2. For example, RAR interacts with CoRNR1, whereas PPARs almost exclusively bind CoRNR2. Here we show that LXRs are capable of interacting with both CoRNR boxes. Consistent with their interactions with corepressors, the LBDs of LXRs repress transcription when fused to the GAL4 DNA binding domain. It is noteworthy that LXR interactions with CoRNR2 are stronger than with CoRNR1, a property that is similar to that of TR. Mutating two homologous residues within LBD that are involved in TR-corepressor interactions also abolished LXR-corepressor interactions (Fig. 3Go). Moreover, LXRs also display a much greater preference for N-CoR over SMRT and cotransfection of N-CoR, but not SMRT, decreases the basal activity of LXREs (Fig. 4BGo), suggesting N-CoR is the major corepressor that binds unliganded LXRs. The corepressor preference by LXR could affect LXR functions in different tissues as N-CoR and SMRT expression patterns do not overlap (34). Our ChIP experiments also showed that N-CoR is recruited to endogenous LXR target gene promoters in the absence of LXR ligand. These data further confirm that corepressors are involved in regulating LXR target gene expression and are likely to be physiologically relevant.

Oxysterols are thought to serve as endogenous LXR ligands and accumulation of cholesterol leads to activation of LXR target genes (35). Our results indicate that LXRs bind corepressors and repress transcription when LXR is not occupied by an agonist ligand. This discovery suggests a model that, in cells when cholesterol (oxysterols) levels are low, LXRs bind to the target gene promoters and actively repress transcription of target genes. For example, unliganded LXR may repress ABCA1 expression, leading to a decreased cholesterol efflux. As a consequence, repression by LXR may be important to regulate cellular cholesterol to steady state levels to support normal cell function. Thus, repression by LXR provides another layer of control in regulating intracellular cholesterol levels. When cholesterol levels are low, LXR would also inhibit SREBP1c and genes involved in fatty acid synthesis, leading to decreased esterification of cholesterols to preserve free cholesterols. When cholesterol levels are high, LXR would release corepressors and instead recruit coactivators, leading to activation of its target genes, such as ABCA1 to pump cholesterol out of cells (Fig. 6Go).



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Figure 6. Model of LXR Function under Different Cellular Cholesterol Levels

Left, When cholesterol levels are low, corepressors are recruited to LXR target genes and as a result, expression of target genes are inhibited. Right, When cellular cholesterol levels are high, corepressors are released and coactivators are recruited, leading to induction of LXR target genes.

 
Our finding further solidifies the role of LXR as a cholesterol sensor. The basal repression together with LXR{alpha} autoregulation provides a very sensitive system for controlling cholesterol levels in human cells. When intracellular cholesterol levels are low, this system decreases the cholesterol efflux pathway to preserve cholesterol. When cholesterol levels are high, this system can respond very quickly to amplify the efflux pathway to remove cholesterol.

The positive effect of LXR agonists on cholesterol efflux would be expected to reduce atherosclerotic risk and recent reports indicate that LXR agonists inhibit atherosclerosis (25, 26). On the other hand, LXR agonists also increase liver SREBP1c expression and triglyceride synthesis, which would have a deleterious effect on metabolic balance. This raises concerns regarding the use of LXRs as potential drug targets. Perhaps a solution to this would be to design selective LXR ligands that activate cholesterol efflux in peripheral tissues but exert a minimal effect on triglyceride synthesis in the liver. Studies on selective estrogen receptor modulators (SERMs) indicate that SERMs can function as agonists in one cell type but as antagonists in others depending on the corepressor levels in the two cell types (36). The discovery that LXRs interact with corepressors provides greater insight into the molecular mechanisms involved in LXR function. In analogy to SERMs, it also suggests that it may be possible to design selective LXR modulators that exploit the intracellular levels of coactivators and corepressors within different cells


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Constructs
LXR mutations were made by site-directed mutagenesis using QuikChange kit (Stratagene, La Jolla, CA). VP-LXR{alpha} and VP-LXRß were constructed by inserting coding sequences of LXR{alpha} and LXRß, respectively, into pACT vector (Promega Corp., Madison, WI). The other constructs were described (10, 37).

Cell Culture and Transfection
The 293 cells were maintained in DMEM plus 10% FBS. Transfection conditions were performed in 96-well as described (37, 38). After transfection, compounds were added in DMEM plus 10% lipoprotein-free FBS (Intracell, Frederick, MD). Cells were harvested 24 h later for measuring luciferase and ß-galactosidase activities.

ALPHA Screen
ALPHA Screen assay is a beads-based, time-resolved amplified luminescent proximity homogeneous assay. GST-LXR{alpha}-LBD expressed in bacterial cells was incubated with 250 nM biotinylated CoRNR1 with and without 10 µM LXR agonist T0901317 overnight at 4 C. An inactive biotinylated peptide was used as a control. The assay was performed in 384-well, 25 µl format in buffer containing 50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 2 mM MgCl2; 1 mM dithiothreitol; 20 nM streptavidin donor beads; and 20 nM anti-GST antibody acceptor beads. The assay plates were read on a Fusion microplate reader (Packard BioScience, Meriden, CT).

Taqman Analysis
RNAs were isolated using QIAGEN (Valencia, CA) RNeasy mini kit. Real-time PCR was performed as described on ABI Prism 7700 using Cyclophilin as a control. Relative expression was determined using the comparative CT method.

ChIP
ChIP experiments were performed according to Manufacturer’s instruction (Upstate Biotechnology, Inc., Lake Placid, NY) with minor modifications. Basically, cells were cross-linked and chromatin templates were broken into approximately 500-bp fragments by sonication. The sonicated fragments were then immunoprecipitated overnight using various antibodies. The LXR antibody was raised in rabbit against a specific peptide (peptide sequence: RAEPPSEPTEIRPQKRKK) in the N-terminal region of LXR{alpha}. This antibody does not cross-react with LXRß. The Flag antibody was purchased from Sigma (St. Louis, MO). The immunoprecipitated complexes were then reverse-cross-linked overnight and eluted. DNA was then purified using QIAGEN PCR purification kit. PCR was done using primers surrounding the LXREs in ABCA1 and SREBP1c promoters. The primers are, for ABCA1, forward, 5' GCGGCTGAACGTCGCCC, reverse, 5'GGGTCGGCTCGGCTCTG; for SREBP1c, forward, 5'TCAGGGTGCCAGCGAACC, reverse, 5'GCTCGAGTTTCACCCCGC.


    ACKNOWLEDGMENTS
 
We thank Dr. Mitchell Lazar for plasmids and Helen Hartman for helpful discussion on chromatin immunoprecipitation experiments. We acknowledge the excellent technical assistance provided by Charles Bolten on Taqman analysis.


    FOOTNOTES
 
Abbreviations: ABCA1, ATP-dependent cholesterol transporter; ALPHA, amplified luminescent proximity homogeneous assay; ChIP, chromatin immunoprecipitation; CoRNR, corepressor nuclear receptor interaction motifs in N-CoR and SMRT; GST, glutathione-S-transferase; LBD, ligand binding domain; LXR, liver X receptor; LXRE, LXR response element; N-CoR, nuclear receptor corepressor; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; SERMs, selective estrogen receptor modulators; SMRT, silent mediator of retinoic acid receptor and thyroid receptor; SREBP1c, sterol response element binding protein; TR, thyroid receptor; VP, viral protein.

Received for publication November 27, 2002. Accepted for publication March 17, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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