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 ...

ASC-2, a multifunctional coactivator, forms a steady-state complex, named ASCOM (for ASC-2 COMplex), that contains the histone H3-lysine-4 (H3K4)-methyltransferase MLL3 or its paralogue MLL4. Somewhat surprisingly, given prior indications of redundancy between MLL3 and MLL4, targeted inactivation of the MLL3 H3K4-methylation activity in mice is found to result in ureter epithelial tumors. Interestingly, this phenotype is exacerbated in a p53 ؉/؊ background and the tumorigenic cells are heavily immunostained for ␥H2AX, indicating a contribution of MLL3 to the DNA damage response pathway through p53. Consistent with the in vivo observations, and the demonstration of a direct interaction between p53 and ASCOM, cell-based assays have revealed that ASCOM, through ASC-2 and MLL3/4, acts as a p53 coactivator and is required for H3K4-trimethyation and expression of endogenous p53-target genes in response to the DNA damaging agent doxorubicin. In support of redundant functions for MLL3 and MLL4 for some events, siRNA-mediated down-regulation of both MLL3 and MLL4 is required to suppress doxorubicin-inducible expression of several p53-target genes. Importantly, this study identifies a specific H3K4 methytransferase complex, ASCOM, as a physiologically relevant coactivator for p53 and implicates ASCOM in the p53 tumor suppression pathway in vivo.

Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in β-cell dysfunction and diabetes. However, it is unknown if this is due to loss of cell-autonomous function of Bmal1 in β cells. To address this, we generated mice with β-cell clock disruption by deleting Bmal1 in β cells (β-Bmal1(-/-)). β-Bmal1(-/-) mice develop diabetes due to loss of glucose-stimulated insulin secretion (GSIS). This loss of GSIS is due to the accumulation of reactive oxygen species (ROS) and consequent mitochondrial uncoupling, as it is fully rescued by scavenging of the ROS or by inhibition of uncoupling protein 2. The expression of the master antioxidant regulatory factor Nrf2 (nuclear factor erythroid 2-related factor 2) and its targets, Sesn2, Prdx3, Gclc, and Gclm, was decreased in β-Bmal1(-/-) islets, which may contribute to the observed increase in ROS accumulation. In addition, by chromatin immunoprecipitation experiments, we show that Nrf2 is a direct transcriptional target of Bmal1. Interestingly, simulation of shift work-induced circadian misalignment in mice recapitulates many of the defects seen in Bmal1-deficient islets. Thus, the cell-autonomous function of Bmal1 is required for normal β-cell function by mitigating oxidative stress and serves to preserve β-cell function in the face of circadian misalignment.

peroxisome proliferator-activated receptor ␥ ͉ transcription ͉ activating signal cointegrator-2 ͉ coactivator ͉ ASCOM

Circadian clocks in adipose tissue are known to regulate adipocyte biology. Although circadian dysregulation is associated with development of obesity, the underlying mechanism has not been established. Here we report that disruption of the clock gene, brain and muscle Arnt-like 1 (Bmal1), in mice led to increased adipogenesis, adipocyte hypertrophy, and obesity, compared to wild-type (WT) mice. This is due to its cell-autonomous effect, as Bmal1 deficiency in embryonic fibroblasts, as well as stable shRNA knockdown (KD) in 3T3-L1 preadipocyte and C3H10T1/2 mesenchymal stem cells, promoted adipogenic differentiation. We demonstrate that attenuation of Bmal1 function resulted in down-regulation of genes in the canonical Wnt pathway, known to suppress adipogenesis. Promoters of these genes (Wnt10a, β-catenin, Dishevelled2, TCF3) displayed Bmal1 occupancy, indicating direct circadian regulation by Bmal1. As a result, Wnt signaling activity was attenuated by Bmal1 KD and augmented by its overexpression. Furthermore, stabilizing β-catenin through Wnt ligand or GSK-3β inhibition achieved partial restoration of blunted Wnt activity and suppression of increased adipogenesis induced by Bmal1 KD. Taken together, our study demonstrates that Bmal1 is a critical negative regulator of adipocyte development through transcriptional control of components of the canonical Wnt signaling cascade, and provides a mechanistic link between circadian disruption and obesity.