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Molecular Endocrinology, doi:10.1210/me.2008-0012
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Molecular Endocrinology 22 (6): 1312-1319
Copyright © 2008 by The Endocrine Society

Activating Signal Cointegrator-2 Is an Essential Adaptor to Recruit Histone H3 Lysine 4 Methyltransferases MLL3 and MLL4 to the Liver X Receptors

Seunghee Lee1, Jeongkyung Lee1, Soo-Kyung Lee and Jae W. Lee

Departments of Molecular and Cellular Biology (S.L., S.-K.L., J.W.L.), Medicine-Division Diabetes, Endocrinology and Metabolism (J.L., J.W.L.), Molecular and Human Genetics (S.-K.L., J.W.L.), Neuroscience (S.-K.L.), The Huffington Center on Aging (S.-K.L.), and Program in Developmental Biology (S.-K.L.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Jae W. Lee, One Baylor Plaza N520.06, Houston, Texas 77030. E-mail: jwlee{at}bcm.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activating signal cointegrator-2 (ASC-2), a coactivator of multiple nuclear receptors and transcription factors, including the liver X receptors (LXRs), is associated with histone H3 lysine 4 (H3K4) methyltransferase (H3K4MT) MLL3 or its paralogue MLL4 in a steady-state complex named ASCOM (ASC-2 complex). ASCOM belongs to Set1-like complexes, a conserved family of related H3K4MT complexes. ASC-2 binds to many nuclear receptors in a ligand-dependent manner through its two LXXLL motifs. In particular, the second motif has been shown to specifically recognize LXRs. However, the exact role for neither ASC-2 nor MLL3/4 in LXR transactivation is clearly defined. Here, we show that the key function of ASC-2 in transactivation by LXRs is to present MLL3 and MLL4 to LXRs. Thus, ASC-2 is required for ligand-induced recruitment of MLL3 and MLL4 to LXRs, and LXR ligand T1317 induces not only expression of LXR-target genes but also their H3K4-trimethylation. Strikingly, both of these ligand effects are ablated in ASC-2-null cells but only partially suppressed in cells expressing an enzymatically inactivated mutant MLL3. Our results also reveal that transactivation by LXRs does not appear to require other Set1-like complexes. Taken together, these results suggest that ASCOM-MLL3 and ASCOM-MLL4 play redundant but essential roles in ligand-dependent H3K4 trimethylation and expression of LXR-target genes, and that ASC-2 is likely a key determinant for LXRs to function through ASCOM but not other Set1-like complexes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS BIND to hormone response elements in target genes and regulate transcription initiation in a ligand-dependent manner (1). Upon ligand binding, the conserved C-terminal ligand binding domain undergoes a structural change (1) that is recognized by an {alpha}-helical LXXLL motif [nuclear receptor (NR) box] of transcriptional coactivators (2). ASC-2 (for activating signal cointegrator-2; also named NCOA6, AIB3, TRBP, RAP250, NRC, and PRIP), a coactivator of many nuclear receptors and transcription factors, contains two NR boxes (3). NR1 binds multiple nuclear receptors, including retinoic acid receptor (RAR) and estrogen receptors (ERs), whereas NR2 specifically interacts with the liver X receptors (LXRs). The physiological importance of ASC-2 as a key coactivator of these nuclear receptors and the pivotal roles of its NR boxes in this context have been proposed from studies with various ASC-2 mouse models (3, 4).

LXR{alpha} (NR1H3) and LXRβ (NR1H2) control cholesterol and fatty acid metabolism by binding as a heterodimer with the retinoid X receptor (RXR) to the LXR-response elements (LXREs) of LXR-target genes (5). Oxysterols such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol (5) that are cholesterol metabolites serve as the physiological ligands for LXRs. Activation of LXR in macrophages results in increased expression of genes encoding the ATP-binding cassette (ABC) cholesterol transporters ABCA1 and ABCG1 as well as apolipoprotein E, which are involved with cholesterol efflux from macrophages toward high-density lipoproteins. In the liver, LXR leads to increased expression of Cyp7A1, encoding a key enzyme in the conversion of cholesterol into bile acids, and ABCG5/ABCG8, which encode ABC transporters implicated in biliary cholesterol excretion. Activation of LXR induces expression of intestinal ABCA1, ABCG5, and ABCG8, thereby accelerating fecal cholesterol disposal by reducing the efficiency of cholesterol absorption. LXR also controls genes that encode proteins involved in de novo lipogenesis. LXR induces the transcription of sterol regulatory element binding protein 1c (SREBP-1c), which encodes a transcription factor that regulates the expression of various lipogenic genes, including those encoding acetyl-coenzyme A carboxylase and fatty acid synthase (FAS). In addition, LXR directly controls the transcription of genes encoding lipoprotein lipase, FAS, stearoyl-coenzyme A desaturase 1, and cholesterol ester transfer protein. Interestingly, D-glucose and D-glucose-6-phosphate have recently been shown as the additional physiological ligands for LXRs in the liver (6), revealing a role for LXRs in a transcriptional switch that links hepatic glucose metabolism to fatty acid synthesis. LXR agonists have been shown to improve glucose tolerance and insulin sensitivity in diabetic animals by increasing expression of glucose transporter 4 and glucose uptake in adipocytes and by suppressing the genes for rate-limiting gluconeogenic enzymes, such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (7, 8, 9).

Histone H3 lysine 4 (H3K4) methylation, an evolutionarily conserved mark for transcriptionally active chromatin, appears to counter the generally repressive chromatin environment imposed by H3-K9/K27 methylation in higher eukaryotes (10). In particular, H3K4 trimethylation is associated with promoters and early transcribed regions of active genes (11, 12). Enzymes for H3K4 methylation include yeast Set1 (ySet1), hSet1, MLL1, MLL2, MLL3/HALR and MLL4/ALR, Ash1, and Set7/9. MLLs and Set1s belong to a family of related complexes (Fig. 1AGo), named Set1-like complexes (13). ASC-2 is an integral component of a Set1-like complex that we named ASCOM (for ASC-2 complex), which contains MLL3, MLL4–1, or MLL4–2 (14, 15). MLL4–1 and MLL4–2 are encoded by the same gene, and they differ only at their N termini (14). The C termini of MLL3 and MLL4 contain a SET domain, known to be associated with an intrinsic histone lysine-specific methyltransferase activity (10). Indeed, we and others have shown that ASCOM is a genuine H3K4-methyltransferase complex (14, 15, 16, 17). More recent studies also identified additional components of ASCOM, including PTIP, PTIP-associated 1, and UTX (16, 17). UTX has been subsequently shown to be a H3K27-demethyase enzyme (18, 19, 20, 21). Thus, ASCOM contains two distinct histone-modifying enzymes linked to transcriptional activation.


Figure 1
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  		Fig. 1. ASC-2 Is Essential for LXR Transactivation

A, Set1-like complexes. Unique subunits in each complex are denoted in bold. B, RT-PCR analysis of E9.5 MEFs from wild-type (wt) and ASC-2–/– mice reveal that LXR-target genes SREBP-1c, ABCA1, and ABCG1 are enhanced in expression by T1317 in wild-type MEFs but not in ASC-2–/– MEFs. ASC-2–/– cells indeed lack ASC-2, as shown. Glyceraldehyde-3-phosphate dehydrogenase represents negative control. Similar results were obtained from three independent experiments. C, Q-PCR using mRNAs in panel A confirms the essentiality of ASC-2 in expression of LXR-target gene, ABCA1. D, HeLa cells stably expressing control siRNA or specific siRNA against ASC-2 (33 ) were immunoblotted with antibodies against ASC-2 and β-actin (control), and monitored for ABCA1 transcripts using Q-PCR. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

 
Our recent results revealed that RAR transactivation is accompanied by RA-induced H3K4 trimethylation and this modification is redundantly mediated by ASCOM-MLL3 and ASCOM-MLL4 (15). We have demonstrated that this is primarily due to the ability of ASC-2 to function as a specific linker for RARs to recruit ASCOM (15). Consistent with this notion, H3K4 trimethylation of RAR-target genes was not affected by related MLL1/2-complexes containing Menin (Fig. 1AGo), encoded by MEN1 (for multiple endocrine neoplasia type 1) tumor suppressor gene (15). These results, along with the fact that ASC-2 was originally isolated as an interacting protein of multiple nuclear receptors (3), suggest that ASCOM is a major H3K4-methyltransferase (H3K4MT) complex for nuclear receptors and that ASC-2 is a key adaptor for nuclear receptors to recruit ASCOM. However, it was recently proposed that direct interactions with MLL4 (22) and Menin (23) are responsible for tethering ER{alpha} to at least three Set1-like complexes during ER{alpha} transactivation; MLL1/2 complexes containing Menin as well as ASCOM-MLL4 (Fig. 1AGo). These results show that other Set1-like complexes are also involved with NR transactivation and ASC-2 is not a sole adaptor for nuclear receptors to recruit ASCOM. Thus, to fully understand the molecular mechanisms by which nuclear receptors accomplish H3K4-trimethylation of their target genes, it is important to determine the identity of Set1-like complex required for each NR and then to identify functional NR adaptor(s) among its subunits. Here we show that ASC-2, similar to its role with RARs (15), tethers ASCOM-MLL3 and ASCOM-MLL4 to LXRs, and that ASCOM, but not MLL1/2 complexes containing Menin, is involved with LXR transactivation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ASC-2 Is an Essential Adaptor for LXRs to Recruit MLL3 and MLL4
Although ASC-2 has been proposed to function as a coactivator of LXRs via its second NR box (4, 24, 25), the loss of function for ASC-2 in LXR transactivation has not been assessed. Thus, we examined the expression of key LXR-target genes in primary mouse embryo fibroblasts (MEFs) isolated from embryonic d 9.5 (E9.5) embryos of wild-type and ASC-2-null mice (26). Whereas the synthetic LXR ligand T1317 further increased expression of SREBP-1c, ABCA1, and ABCG1 in wild-type MEFs, their expression was completely abolished both in the absence and presence of T1317 in ASC-2–/– MEFs (Fig. 1BGo). These results were confirmed for ABCA1 transcripts in quantitative RT-PCR (Q-PCR) experiments (Fig. 1CGo). We obtained similar results for ABCA1 transcripts from HeLa cells the expression of which for ASC-2 was compromised using specific short interfering RNA (siRNA) (Fig. 1DGo). These results clearly demonstrate the essentiality of ASC-2 in LXR transactivation.

Next, we wished to examine the role of ASCOM in H3K4-methylation of LXR-target genes using chromatin immunoprecipitation (ChIP) assays. Because ASC-2-null embryos die at E9.5, however, it was difficult to isolate a sufficient amount of MEFs from ASC-2-null embryos. Thus, we decided to utilize the previously described cell lines derived from E9.5 wild-type and ASC-2–/– MEFs (15). To test whether these cells retain intact LXR-signaling pathways, we examined the expression pattern of LXR-target genes and the recruitment pattern of ASC-2 to the LXR response elements (LXREs) in ABCA1. In wild-type cells, T1317 efficiently induced expression of SREBP-1c and ABCA1 (data not shown) and supported transactivation of a luciferase reporter containing LXREs (Fig. 2AGo). Consistent with the above expression study (Fig. 1Go, B–D), ASC-2–/– cells failed to support transactivation of this reporter, whereas the normal responsiveness to LXR and T1317 was restored by reexpression of ASC-2 (Fig. 2AGo). In addition, in wild-type cells, ASC-2 was recruited to ABCA1-LXREs and this binding was significantly enhanced by treatment with T1317 (Fig. 2BGo). It should be noted that the weaker recruitment of ASC-2 without T1317 (Fig. 2BGo) is consistent with the fact that LXR remains partially activated due to the presence of a basal level of endogenous ligands (5). Consistent with the stable association of ASC-2 with either MLL3 or MLL4 in a steady-state complex ASCOM (15), wild-type cells were observed to have a significant level of H3K4-trimethylation in ABCA1-LXREs even in the absence of T1317, which was substantially increased in the presence of T1317 (Fig. 2CGo). Remarkably, both the basal and T1317-induced levels of H3K4-trimethylation of ABCA1-LXREs were ablated in ASC-2–/– cells (Fig. 2CGo). In contrast, ASC-2–/– cells still supported H3K4 trimethylation of Hoxa9, a target gene of MLL1 Set1-like complex (27, 28). These results suggest that, during LXR transactivation, ASC-2 presents MLL3 and/or MLL4 to LXRs, thereby enabling H3K4-trimethylation of LXR-target genes. In direct support of this idea, whereas T1317 enhanced recruitment of both MLL3 and MLL4 to ABCA1-LXREs in wild-type cells, their recruitment to ABCA1-LXREs was abolished both in the absence and presence of T1317 in ASC2–/– cells, although MLL3 and MLL4 were equally expressed in both cell types (Fig. 2DGo). Thus, we concluded that ASC-2 is an essential adaptor for LXRs to recruit MLL3 and MLL4 during LXR transactivation.


Figure 2
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  		Fig. 2. ASC-2 Is Essential for H3K4-Trimethylation of LXR-Target Genes

A, Transfection assays for LXRE:LUC reporter in wild-type (wt) and ASC-2–/– cells established from E9.5 MEFs (15 ). B, ChIP assays using anti-ASC-2 antibody reveal that ASC-2 is recruited to the LXREs of ABCA1 in wild-type MEFs. Similar results were obtained in HEK293, HepG2, and HeLa cells (data not shown). C, ChIP assays using antitrimethylated H3K4 demonstrate that T1317-enhanced H3K4-trimethylation of ABCA1-LXREs is observed in wild-type but not in ASC-2–/– MEFs. D, ChIP assays using anti-MLL3 and MLL4 antibodies show that ASC-2–/– cells no longer support the T1317-enhanced recruitment of MLL3/4 to ABCA1-LXREs, which is observed readily in wild-type cells. MLL3 and MLL4 were equally expressed in both cell types, as measured by Q-PCR. Inputs indicate 2% of the reactions (B–D).

 
Redundant Function of MLL3 and MLL4 during LXR Transactivation
Our results suggest that ASC-2 is a key linker that connects ASCOM and LXRs. Interestingly, a complex similar to ASCOM-MLL4 has been isolated from an ER-negative breast cancer cell line DU4475, based on its estradiol-dependent interaction with GST-ER{alpha} (22). In addition, ER{alpha} was found to directly bind to two NR boxes in the C-terminal region of MLL4 in an estradiol-dependent manner and siRNA-mediated down-regulation of MLL4 alone significantly inhibited ER{alpha} transactivation (22). Based on these results, they proposed that MLL4 functions as a direct adaptor for ER{alpha} to recruit MLL4 complex, which plays crucial roles in ER{alpha} signaling (22). These results challenge the proposed universal adaptor role for ASC-2 to link ASCOM to ASC-2-interacting nuclear receptors (3, 15). These results are also in sharp contrast to our results demonstrating the requirement for reduced expression for both MLL3 and MLL4 to impair RAR transactivation (15). Moreover, MLL1/2 complexes (Fig. 1AGo) have also been shown to be tethered to ER{alpha} via an adaptor function of Menin, which directly interacts with ER{alpha} (23). These intriguing results led us to examine whether MLL3 and/or MLL4 also function as alternative and/or additional adaptors to recruit ASCOM to LXRs, along with the interactions of ASC-2 and LXRs (24), and whether Menin can directly interact with LXRs, thereby recruiting Menin-MLL1/2 complexes to LXRs. Sequence analysis revealed that MLL3 and MLL4 have five and six potential NR boxes, respectively (Fig. 3AGo). As reported (22), ER{alpha} indeed showed strong interaction with MLL4-NR5 in the yeast two-hybrid assays (Fig. 3AGo). Under this condition, the weak interactions of ER{alpha} and MLL4-NR6 (22) were not readily observed. Unexpectedly, we found that NR1 in MLL4 and NR1 in MLL3 also interact with ER{alpha} (Fig. 3AGo), suggesting that MLL3 may also play roles in ER{alpha} signaling via direct interactions with ER{alpha}. It should be noted, however, that the in vivo functionality of these interactions remains to be tested. Importantly, none of the NR motifs in MLL3 and MLL4 interacted with RAR{alpha} and LXR{alpha} (Fig. 3AGo). In addition, both wild-type and Menin–/– MEFs nicely supported T1317-enhanced expression of ABCG1, a direct LXR-target gene (Fig. 3BGo). Taken together, these results led us to conclude that transactivation by RARs (15) and LXRs (Fig. 2AGo) specifically requires ASCOM, unlike the involvement of multiple Set1-like complexes with ER{alpha}. These results also reinforce the idea that ASC-2 is a major adaptor to link both RARs and LXRs to ASCOM.


Figure 3
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  		Fig. 3. LXR Specifically Requires ASCOM But Not Other Set1-Like Complexes

A, Sequences of five and six potential NR boxes found in MLL3 and MLL4, respectively. In the yeast two-hybrid assays, ER{alpha} interacts with MLL3-NR1, MLL4-NR1, and MLL4-NR5. In contrast, RAR{alpha} and LXR{alpha} interact with none of these NR boxes. B, Expression of ABCG1 was readily enhanced by T1317 in both wild-type and Menin–/– MEFs, indicating that Menin-containing MLL1/2 H3K3MT complexes are not essential for LXR transactivation. C, HepG2 cells were transfected with control siRNA, siRNA against MLL3 or MLL4, or siRNAs for both MLL3 and MLL4. Cells were treated 2 d after transfection with vehicle or 10 µM T1317 for 12 h, and they were then tested for FAS transcript levels by Q-PCR. Similar results were obtained in three independent experiments. con, Control; E2, estradiol.

 
To further test whether MLL3 and MLL4 act redundantly with LXRs, we used the previously defined siRNAs directed against human MLL3 and MLL4 (15). These siRNAs are highly specific, because they affect neither each other’s expression nor expression of multiple other genes that we have tested, including ASC-2 and glyceraldehyde-3-phosphate dehydrogenase (15). Due to the inefficiency of MEFs in transfection, we have not yet developed siRNAs against mouse MLL3 and MLL4. Thus, we expressed these siRNAs in human HepG2 cells. Remarkably, T1317-induced expression of LXR-target genes FAS, SREBP-1c, and ABCA1 was negligibly affected by siMLL3 or siMLL4 alone but significantly impaired with simultaneous transfection of both siRNAs (Fig. 3CGo and data not shown). These results clearly demonstrate that MLL3 and MLL4 play redundant roles in transactivation by LXRs, similar to their roles with RARs (15).

Partial Induction of LXR-Target Genes and Their H3K4-Trimethylation in MLL3{Delta}/{Delta} Mice
To study the role of MLL3 in NR transactivation, we have recently established a mutant mouse line with a small deletion in the MLL3-SET domain that inactivates the enzymatic activity of MLL3 (15). Wild-type mice routinely develop fatty liver when they are fed a high-fat diet. Interestingly, however, this phenotype was not evident in the homozygous MLL3{Delta}/{Delta} mutant mice (Fig. 4AGo). Staining with hematoxylin and eosin and oil-red-O revealed that MLL3{Delta}/{Delta} mice indeed did not accumulate lipid droplets as much as their wild-type control mice (Fig. 4AGo). Given the potential multifunctionality of MLL3, these phenotypes may result from complex defects involving multiple target transcription factors of MLL3. Nonetheless, we reasoned that these results may result, at least in part, from a possible role for MLL3 in regulating expression of hepatic de novo lipogenic genes directed by LXRs. In support of this idea, analysis of hepatic mRNAs isolated from these animals revealed that LXR-target genes in lipogenesis, FAS and SREBP-1c, were significantly suppressed in the livers of MLL3{Delta}/{Delta} mice both on normal chow and high-fat diets (Fig. 4BGo). In addition, directly supporting the role for MLL3 in regulating hepatic lipogenic target genes of LXRs, T1317-induced expression of ABCA1, SREBP-1c, and FAS was impaired in primary MEFs isolated from E13.5 MLL3{Delta}/{Delta} embryos (Fig. 4CGo). It is important to note that these genes were still significantly induced by T1317 (Fig. 4CGo), suggesting that another H3K4MT, most likely MLL4, still functions in these cells. In sharp contrast, their expression was almost entirely ablated in ASC-2–/– MEFs (Fig. 1Go, B–D). Taken together, these results suggest that ASCOM-MLL3 plays crucial roles in LXR transactivation and that ASCOM-MLL3 and ASCOM-MLL4 are likely to function redundantly. In further support of this idea, the basal and T1317-induced level of H3K4-trimethylation of ABCA1-LXREs was relatively weakly but reproducibly observed in primary MLL3{Delta}/{Delta} MEFs (Fig. 4DGo). It should be noted that we have previously shown that ASC-2 and MLL4 are comparably expressed in wild-type and MLL3{Delta}/{Delta} MEFs (15). In contrast, these cells readily supported H3K4 trimethylation of Hoxa9, a target of MLL1 Set1-like complex (27, 28). These results, along with our finding that H3K4-trimethylation of ABCA1-LXREs was ablated in ASC-2–/– cells (Fig. 2CGo), strongly suggest that MLL3 and MLL4 function as redundant H3K4MTs for LXR-target genes.


Figure 4
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  		Fig. 4. MLL3 Is Involved with Expression of LXR-Target Genes

A, The liver sections from 2-month-old wild-type (wt) and MLL3{Delta}/{Delta} male mice (n = 3~5) fed normal chow diet or high-fat diet for 2 months were analyzed with hematoxylin and eosin and oil-red-O staining. All MLL3{Delta}/{Delta} mice accumulated less lipid droplets than wild-type mice. A set of representative images is shown. B, Analyses of total mRNA isolated from these mice by Q-PCR show that FAS and SREBP-1c are significantly down-regulated in MLL3{Delta}/{Delta} mice under both conditions. Values are the mean ± SE (*, P < 0.05; **, P < 0.01). C, T1317-enhanced expression of ABCA1, SREBP-1c, and FAS was only partially impaired in E13.5 MLL3{Delta}/{Delta} MEFs. Similar results were obtained in two independent experiments. D, ChIP assays using antitrimethylated H3K4 demonstrate that both basal and T1317-enhanced level of H3K4-trimethylation of ABCA1-LXREs is significantly reduced, but still present, in E13.5 MLL3{Delta}/{Delta} MEFs in comparison with E13.5 wild-type MEFs. In contrast, H3K4-trimethylation of Hoxa9 was readily detected in both cell types. Inputs indicate 2% of the reactions. H&E, Hematoxylin and eosin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
H3K4-trimethylation is an evolutionarily highly conserved mark for active transcription (11, 12). In contrast to a single enzyme responsible for this modification in yeast, mammals have multiple H3K4MTs, including a family of steady-state complexes named Set1-like complexes (13). This family includes ASCOM, which contains ASC-2, a coactivator for many members of the NR superfamily (3). Thus, this complex has been suggested as a major H3K4MT complex for nuclear receptors (14, 15). However, given the involvement of at least three Set1-like complexes with ER{alpha} (22, 23), this is apparently an overly simplified view, and it is important to carefully define the identity of the H3K4MT complex required for each nuclear receptor. In this study, we addressed this issue for LXRs and provided strong evidence that 1) LXR transactivation is accompanied by H3K4 trimethylation of their target genes and this methylation is carried out redundantly by ASCOM-MLL3 and ASCOM-MLL4, and 2) that the recruitment of these two complexes to LXRs depends on ASC-2. This requirement for ASC-2 in recruiting ASCOM to LXRs is expected to reflect the ability of ASC-2-NR2 to interact with LXRs in a ligand-induced manner (4, 24, 25). Although RXR can bind to ASC-2-NR1 (3), we have recently found that RXR allosterically enhances LXR binding to ASC-2-NR2 without directly binding to ASC-2-NR1 and consistently ASC-2-NR2, but not ASC-2-NR1, is essential for LXR transactivation (29). Overall, these results led to a model (Fig. 5Go), in which ASC-2 plays key roles in triggering H3K4 trimethylation of LXR-target genes due to the ability of ASC-2-NR2 to tether ASCOM to the LXR-RXR heterodimer bound to LXR-target genes in a ligand-dependent manner.


Figure 5
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  		Fig. 5. The Working Model

This study, along with our previous results (24 25 29 ), suggest that ASC-2 tethers ASCOM-MLL3 and ASCOM-MLL4 to LXR-RXR heterodimer through ligand-dependent direct interactions of ASC-2-NR2 and LXR. This, in turn, leads to ligand-dependent H3K4 trimethylation (me) of LXR-target genes. Importantly, expression of LXR-target genes is correlated to their H3K4 trimethylation, supporting the important role for this histone H3 modification in LXR transactivation.

 
We have recently proposed that a subunit of Set1-like complexes determines the target specificity of each complex via direct protein-protein interactions with target transcription factors (15). The current study extends this notion by validating the previously proposed specialized function for ASC-2 as a key adaptor to present ASCOM to LXRs (4, 15, 24, 25). This is most evident from our experimental results that T1317-enhanced recruitment of both MLL3 and MLL4 to LXRs and T1317-enhanced H3K4-trimentylation of LXR-target gene ABCA1 were abolished in ASC-2-null cells (Fig. 2Go, C and D). Further support for our model (Fig. 5Go) is provided by our results that MLL3{Delta}/{Delta} MEFs were only partially impaired in the ability to support T1317-induced expression of LXR-target genes and their T1317-enhanced H3K4-trimethylation (Fig. 4Go, C and D). The LXR specificity of ASCOM is also supported by our finding that, whereas ASC-2 was required for LXR transactivation, an integral component of MLL1/2 Set1-like complexes, Menin, was dispensable (Fig. 3BGo). In comparison, targeted deletion of Menin or siRNA-mediated reduction of MLL4 alone significantly impaired ER{alpha} transactivation (22, 23).

Interestingly, our analysis of hepatic mRNAs revealed that whereas the hepatic lipogenic target genes of LXRs, FAS and SREBP-1c, were suppressed in MLL3{Delta}/{Delta} mice, expression of another direct target gene of LXRs, Cyp7A1, which encodes a key enzyme in the conversion of cholesterol into bile acids, was unaffected (data not shown). These results suggest that clearance of cholesterol via bile acid synthesis could be intact in MLL3{Delta}/{Delta} mice, which, along with the impaired expression of lipogenic genes (Fig. 4Go, B and C), might have contributed to our failure to observe accumulation of oil-red-O-positive lipid droplets in the livers of MLL3{Delta}/{Delta} mice even with a high-fat diet (Fig. 4AGo). Thus, these results raise an interesting possibility that MLL3 may have some selectivity toward a subset of LXR-target genes. Future study should be directed at examining whether MLL3 indeed controls only a selective subset of LXR-target genes, such as those involved in hepatic de novo lipogenesis.

In summary, we have shown that ASC-2 is an essential linker for LXRs to recruit ASCOM-MLL3 and ASCOM-MLL4 to LXRs, and that these two complexes are redundant but essential and specific H3K4MT complexes for LXR-target genes. Importantly, our results also expand the role of H3K4-trimethylation, which has been reported to be tightly coupled to transcriptionally active genes (10, 11, 12), i.e. to genes involved in metabolic homeostasis as well. Moreover, our results, together with the recent discovery of specific H3K4-demthylases (30, 31, 32), raise an exciting possibility that we may find far more dynamic regulation of this modification in numerous metabolic genes targeted by LXRs and other transcription factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
The reporters LexA-LacZ and LXRE:LUC, the transfection indicator pRSV-β-gal, the mammalian expression vectors for ASC-2 and LXR{alpha}, and the yeast two-hybrid vectors encoding LexA, B42, B42-hER{alpha}, and B42-RAR{alpha} were as described elsewhere (24). A yeast expression vector encoding LexA fused to the full-length LXR{alpha} was a gift from Dr. Young-Chul Lee (Chonnam National University, Korea). PCR fragments encoding MLL3-NR1-5 and MLL4-NR1-6 were cloned into the LexA-fusion vector pEG202.

Reverse Transcriptase- and Quantitative-RT-PCR (Q-PCR)
Total RNA was isolated from indicated cell lines or mouse hepatocytes after lysis in TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA), and RT-PCRs were performed as described previously (33). For the SYBR Green Q-PCR, 250 ng of cDNA was used per reaction. Each 25-µl SYBR Green reaction consisted of 5 µl of cDNA (50 ng/µl), 12.5 µl of 2x Universal SYBR Green PCR Master Mix (PE Biosystems, Foster City, CA), and 3.75 µl of 50 nM forward and reverse primers. Optimization was performed for each gene-specific primer before the experiment to confirm that 50 nM primer concentrations did not produce nonspecific primer-dimer amplification signal in no-template control tubes. Q-PCR was performed on ABI5700 PCR Instrument (Applied Biosystems, Foster City, CA) by using three-stage program parameters provided by the manufacturer: 2 min at 50 C, 10 min at 95 C, and then 40 cycles of 15 sec at 95 C, and 1 min at 60 C. Specificity of the amplified product was confirmed by examination of dissociation reaction plots. A distinct single peak indicated that single DNA sequence was amplified during PCR. In addition, appearance of a single, specific band of the correct size was confirmed on ethidium bromide-stained agarose gels. Each sample was tested in triplicate by Q-PCR. Cyclophilin A mRNA levels were used as a reference standard. The primer sequences were as described elsewhere (25).

Transfections
Cells were grown in 24-well plates with medium supplemented with 10% fetal calf serum for 24 h and transfected with 100 ng of LacZ expression vector pRSV-β-gal and 100 ng of LXRE:LUC, along with mammalian expression vectors for LXR{alpha} and ASC-2. Total amounts of expression vectors were kept constant by adding pcDNA3. Transfections and luciferase assays were done in triplicate as described (24), and the results were normalized to the LacZ expression. Similar results were obtained in two independent experiments.

ChIP
Cells were grown in 10-cm dishes with medium supplemented with 10% fetal bovine serum for 24 h. ChIP assays were carried out as described (14, 15) using antibodies against ASC-2, MLL3, and MLL4 (15) and antibody against trimethylated H3K4 (Abcam, Cambridge, MA). The primer sequences for the ABCA1 region encompassing its LXREs were GCT TTC TGC TGA GTG ACT GAA CTA C and GAA TTA CTG CTT TTT GCC GCG. The primer sequences for the Hoxa9 locus were GGT GCG CTC TCC TTC GCG GGC TTA and CAT TCT CGG CAT TGT TTT CGG AGA. Each experiment, repeated more than three times, produced similar results.

Yeast Two-Hybrid Assays and Histology
Yeast two-hybrid assays were done as described previously (24). The mouse livers were excised, frozen, sectioned in 10-µm-thick slices, and stained with hematoxylin and eosin and oil-red-O, as described (25).


   ACKNOWLEDGMENTS
 
We thank Inwon Na, Keuhee Park, Dong-Kee Lee, and Young Hwa Goo for the initial contributions to this work.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK064678 (to J.W.L.).

Disclosure statement: The authors have nothing to declare.

First Published Online March 27, 2008

1 S.L and J.L. contributed equally to this paper and should both be considered first authors. Back

Abbreviations: ABC, ATP-binding cassette; ASC-2, activating signal cointegrator-2; ASCOM, ASC-2 complex; ChIP, chromatin immunoprecipitation; E9.5, embryonic d 9.5; ER, estrogen receptor; FAS, fatty acid synthase; H3K4, histone H3 lysine 4; H3K4MT, histone H3 lysine 4 methyltransferase; LXR, liver X receptor; MEF, mouse embryo fibroblast; NR, nuclear receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; Q-PCR, quantitative-RT-PCR; siRNA, short interfering RNA; SREBP, sterol regulatory element-binding protein.

Received for publication January 10, 2008. Accepted for publication March 17, 2008.


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 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   RARα  |  LXRβ  |  LXRα  |  ERα
Coregulators:   ASC-2
Ligands:   T0901317



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