Activating signal cointegrator-2 (ASC-2), a coactivator of multiple transcription factors that include retinoic acid receptor (RAR), associates with histone H3-K4 methyltranferases (H3K4MTs) MLL3 and MLL4 in mixed-lineage leukemia. Here, we show that mice expressing a SET domain mutant of MLL3 share phenotypes with isogenic ASC2 ؉/؊ mice and that expression and H3-K4 trimethylation of RAR target gene RAR-␤2 are impaired in ASC-2-null mouse embryo fibroblasts (MEFs) or in MEFs expressing siRNAs against both MLL3 and MLL4. We also show that MLL3 and MLL4 are found in distinct ASC-2-containing complexes rather than in a common ASC-2 complex, and they are recruited to RAR-␤2 by ASC-2. In contrast, RAR-␤2 expression is intact in MEFs devoid of menin, a component of MLL1 and MLL2 H3K4MT complexes. These results suggest that ASC-2 confers target gene specificity to MLL3 and MLL4 H3K4MT complexes and that recruitment of H3K4MTs to their target genes generally involves interactions between integral components of H3K4MT complexes and transcription factors.mixed-lineage leukemia (MLL) ͉ retinoic acid receptor ͉ transcription N uclear receptors (NRs) bind hormone response elements in target genes and regulate transcriptional initiation in a liganddependent manner (1). During ligand binding, the conserved C-terminal activation function 2 domain undergoes a structural change (1) that is recognized by an ␣-helical LXXLL motif (NR box) in transcriptional coactivators (2). Activating signal cointegrator-2 (ASC-2; also named AIB3, TRBP, TRAP250, NRC, and PRIP), a coactivator of many NRs and other transcription factors, contains two NR boxes (3). NR box 1 binds multiple NRs, including retinoic acid receptor (RAR), whereas NR box 2 interacts with liver X receptors. The physiological importance of ASC-2 as a key coactivator of these NRs and the pivotal roles of both NR boxes in this context have been proposed from recent studies with various ASC-2 mouse models (3).In HeLa nuclei, ASC-2 resides in a steady-state complex [ASC-2 complex (ASCOM)] (4) that contains retinoblastomabinding protein RbBP5, ␣͞␤-tubulins, and trithorax group proteins Ash2L, MLL4-1͞ALR-1, MLL4-2͞ALR-2, and MLL3͞ HALR (the paralog of MLL4͞ARL). † † MLL4-1 and MLL4-2 (collectively MLL4s) are encoded by the same gene, and they differ only at their N termini (4). The C termini of MLL3 and MLL4s contain a SET domain (5) with an intrinsic histone lysine-specific methyltransferase activity. Indeed, recombinant MLL3 and MLL4 SET domains and partially immunopurified ASCOM exhibit weak but specific histone H3-K4 methyltransferase (H3K4MT) activity in vitro (4).H3-K4 methylation, an evolutionarily conserved mark linked to transcriptionally active chromatin, has been proposed to counter the generally repressive chromatin environment imposed by H3-K9͞K27 methylation in higher eukaryotes (6). In particular, H3-K4 trimethylation is associated with promoters and early transcribed regions of active genes (7,8). H3K4MTs include yeast Set1 (ySet1), hSet1, MLL1, MLL2, and MLL3 and ...

Many transcription coactivators interact with nuclear receptors in a ligandThe nuclear receptor superfamily is a group of proteins that regulate, in a ligand-dependent manner, transcriptional initiation of target genes by binding to specific DNA sequences named hormone response elements (reviewed in reference 23). Functional analysis of nuclear receptors has shown that there are two major activation domains. The N-terminal domain (AF1) contains a ligand-independent activation function, whereas the ligand-binding domain (LBD) exhibits ligand-dependent transactivation function (AF2). The AF2 core region, located at the extreme C terminus of the receptor LBDs, is conserved among nuclear receptors and undergoes a major conformational change upon ligand binding (23). This region has been shown to play a critical role in mediating transactivation by serving as a ligand-dependent interaction interface with many different coactivators (reviewed in reference 9). These coactivators, including the p160 family members (i.e., SRC-1, SRC-2/GRIP1/TIF2, and SRC-3/ACTR/pCIP/AIB1/ RAC3/TRAM1), CBP/p300, p/CAF, TRAP/DRIP, activating signal cointegrator 2 (ASC-2), and many others, bridge nuclear receptors and the basal transcription apparatus and/or remodel the chromatin structures (9).Chromatin, the physiological template of all eukaryotic genetic information, undergoes a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA (reviewed in reference 12). SRC-1 and the p160 family member ACTR, along with CBP and p300, were recently shown to contain histone acetyltransferase (HAT) activities and associate with yet another HAT protein, p/CAF (9). In contrast, SMRT and N-CoR, nuclear receptor corepressors, form complexes with Sin3 and histone deacetylase proteins (9). These results are consistent with the notion that the acetylation of histones destabilizes nucleosomes and relieves transcriptional repression by allowing transcription factors to access recognition elements, whereas deacetylation of the histones stabilizes the repressed state. More recently, the histone arginine methyltransferases CARM1 and PRMT1 were newly defined as transcriptional coactivators of nuclear receptors (4, 40). NSD1 and

The methylation of lysine residues of histones plays a pivotal role in the regulation of chromatin structure and gene expression. Here, we report two crystal structures of SET7/9, a histone methyltransferase (HMTase) that transfers methyl groups to Lys4 of histone H3, in complex with S-adenosyl-L-methionine (AdoMet) determined at 1.7 and 2.3 A Ê resolution. The structures reveal an active site consisting of: (i) a binding pocket between the SET domain and a c-SET helix where an AdoMet molecule in an unusual conformation binds; (ii) a narrow substrate-speci®c channel that only unmethylated lysine residues can access; and (iii) a catalytic tyrosine residue. The methyl group of AdoMet is directed to the narrow channel where a substrate lysine enters from the opposite side. We demonstrate that SET7/9 can transfer two but not three methyl groups to unmodi®ed Lys4 of H3 without substrate dissociation. The unusual features of the SET domain-containing HMTase discriminate between the un-and methylated lysine substrate, and the methylation sites for the histone H3 tail. Keywords: compact form of AdoMet/9 histone methyltransferase/post-SET helix/SET7/SET domain/ substrate-speci®c channel

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Activating signal cointegrator 2 (ASC-2), a cancer-amplified transcriptional coactivator of nuclear receptors and many other transcription factors, contains two LXXLL-type nuclear receptor interaction domains. Interestingly, the second LXXLL motif is highly specific to the liver X receptors (LXRs). In cotransfection, DN2, an ASC-2 fragment encompassing this motif, exerts a potent dominant-negative effect on transactivation by LXRs, which is rescued by ectopic coexpression of the full-length ASC-2 but not by other LXXLL-type coactivators, such as SRC-1 and TRAP220. In contrast, DN2/m, in which the LXXLL motif is mutated to LXXAA to abolish the interactions with LXRs, is without any effect. Accordingly, expression of DN2, but not DN2/m, in transgenic mice results in phenotypes that are highly homologous to those previously observed with LXR␣ ؊/؊ mice, including a rapid accumulation of large amounts of cholesterol and down-regulation of the known lipidmetabolizing target genes of LXR␣ in the liver upon being fed a high-cholesterol diet. These results identify ASC-2 as a physiologically important transcriptional coactivator of LXRs and demonstrate its pivotal role in the liver lipid metabolism.

Daily oscillations in mRNA levels are a general feature of most clock genes. Although mRNA oscillations largely depend on transcriptional regulation, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations in Drosophila. Currently, however, there is no direct evidence for post-transcriptional regulation of mammalian clock genes. To investigate the roles of post-transcriptional regulations, we focused on the 3 -untranslated region (3 -UTR) of mouse Period3 (mPer3) mRNA, one of the clock genes. Insertion of the entire mPer3 3 -UTR downstream of a reporter gene resulted in a dramatic decrease in mRNA stability. Deletion and point mutation analyses led to the identification of critical sequences responsible for mRNA decay. To explore the effects of the mPer3 3 -UTR-mediated mRNA decay on circadian oscillations, we established NIH3T3 stable cell lines that express luciferase mRNA with wild-type or mutant mPer3 3 -UTR. Interestingly, a stabilizing mutation of 3 -UTR induced a significant alteration in the oscillation profile of luciferase mRNA. Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the luciferase mRNA level with mutant 3 -UTR began to increase earlier than that in the presence of wild-type 3 -UTR. Consequently, luciferase mRNA with mutant 3 -UTR displayed oscillation patterns with a prolonged rising phase. Our results indicate that mPer3 3 -UTR-mediated mRNA decay plays an essential role in mRNA cycling and provide direct evidence for post-transcriptional control of circadian mRNA oscillations.Circadian rhythms, defined as daily behavioral and physiological oscillations, have been observed in a variety of organisms. These oscillations are driven by a self-sustained timekeeping system, the intracellular clock (1, 2). Over the past several years, great effort has been focused on understanding the molecular mechanisms that underlie the circadian clock and indispensable components, called clock genes, have been elucidated (3-8). In addition, it was revealed that the intracellular molecular clock consists of interacting positive and negative transcriptional and translational feedback loops that involve these components (9 -12).Daily oscillations in protein and/or mRNA levels are central features of clock genes (1, 12). As for the underlying mechanism of mRNA cycling, a number of studies have revealed that mRNA oscillations largely depend on transcriptional regulation (10, 13-17). In Drosophila, nevertheless, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations. First, despite the absence of rhythmic transcription, period (per) mRNA showed circadian cycling in transgenic flies carrying promoter-less per gene (18). Second, results of a nuclear run-on assay suggested that post-transcriptional regulation is responsible for mRNA cycling (19).In mammals, however, much less is known about the relevance of post-transcriptional control to the circadian oscillation...

For the orphan nuclear receptor subfamily that includes Nur77 (NGFI-B), Nurr1, and NOR-1, no transcriptional coregulators have been identified thus far. In this report, we found that Ca 2؉ /calmodulin-dependent protein kinase IV enhances Nur77 transactivation in cotransfections either alone or in synergy with AF2-dependent coactivator ASC-2, whereas corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) is repressive. Interestingly, Nur77 interacted with SMRT but did not directly bind ASC-2, and accordingly, the putative AF2 core domain of Nur77 did not affect the Nur77 transactivation. SMRT harbors transferable repression domains that associate with various histone deacetylases. Surprisingly, histone deacetylase inhibitor trichostatin A was unable to block the repressive effect of SMRT while dramatically stimulating the Nur77 transactivation. These results suggest that SMRT and ASC-2 are specific coregulators of Nur77 and that SMRT may dynamically compete with a putative adaptor molecule, which links ASC-2 to Nur77, for the identical binding sites within Nur77 in vivo.The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (for a review, see Ref.1). The superfamily includes receptors for a variety of small hydrophobic ligands, such as steroids, T3, and retinoids, as well as a large number of related proteins that do not have known ligands referred to as orphan nuclear receptors. Functional analysis of nuclear receptors has shown that the ligand-binding domain (LBD) 1 exhibits ligand-dependent transcriptional activation function referred to as activation function-2 (AF2). Notably, this region has been shown to play a critical role in mediating transactivation by serving as a ligand-dependent interaction interface with many different coactivators (for reviews, see Refs. 2 and 3). Transcriptional coactivators either bridge transcription factors and the components of the basal transcriptional apparatus and/or remodel the chromatin structures. In particular, cAMP-response element-binding protein (CREB)-binding protein (CBP) and its functional homologue p300, steroid receptor coactivator-1 (SRC-1) and its family members, and activating signal cointegrator-2 (ASC-2) were shown to be essential for the activation of transcription by a large number of regulated transcription factors, including many members of the nuclear receptor superfamily (2, 3). Interestingly, SRC-1 and its family member activator of thyroid and retinoic acid receptors (ACTR) along with CBP and p300 were recently shown to contain histone acetyltransferase activities and associate with another histone acetyltransferase protein p300/CBP-associated factor (P/CAF) (2, 3). In contrast, nuclear receptor corepressor (N-CoR) and its homologue silencing mediator of retinoid and thyroid hormone receptors (SMRT) harbor transferable repression domains that can associate with va...

The daily oscillations observed in most living organisms are endogenously generated with a period of 24 h, and the underlying structure of periodic oscillation is an autoregulatory transcription-translation feedback loop. The mechanisms of untranslated region (UTR)-mediated post-transcriptional regulation (e.g., mRNA degradation and internal ribosomal entry site (IRES)-mediated translation) have been suggested to finetune the expression of clock genes. Mouse Period3 (mPer3) is one of the paralogs of Period gene and its function is important in peripheral clocks and sleep physiology. mPer3 mRNA displays a circadian oscillation as well as a circadian phasedependent stability, while the stability regulators still remain unknown. In this study, we identify three proteins -heterogeneous nuclear ribonucleoprotein (hnRNP) K, polypyrimidine tract-binding protein (PTB), and hnRNP D -that bind to mPer3 mRNA 3 0 -UTR. We show that hnRNP K is a stabilizer that increases the amplitude of circadian mPer3 mRNA oscillation and hnRNP D is a destabilizer that decreases it, while PTB exhibits no effect on mPer3 mRNA expression. Our experiments describe their cytoplasmic roles for the mRNA stability regulation and the circadian amplitude formation. Moreover, our mathematical model suggests a mechanism through which post-transcriptional mRNA stability modulation provides not only the flexibility of oscillation amplitude, but also the robustness of the period and the phase for circadian mPer3 expression.