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.

Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2), a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone–Indian hedgehog–Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.

In several organ systems the transitional zone between different types of epithelia is a hotspot for pre-neoplastic metaplasia and malignancy1–3. However, the cell-of-origin for the metaplastic epithelium and subsequent malignancy, remains obscure1–3. In the case of Barrett’s oesophagus (BE), intestinal metaplasia occurs at the gastro-oesophageal junction, where stratified squamous epithelium transitions into simple columnar cells4. Based on different experimental models, several alternative cell types have been proposed as the source of the metaplasia, but in all cases the evidence is inconclusive and no model completely mimics BE with the presence of intestinal goblet cells5–8. Here, we describe a novel transitional columnar epithelium with distinct basal progenitor cells (p63+ KRT5+ KRT7+) in the squamous-columnar junction (SCJ) in the upper gastrointestinal tract of the mouse. We use multiple models and lineage tracing strategies to show that this unique SCJ basal cell population serves as a source of progenitors for the transitional epithelium. Moreover, upon ectopic expression of CDX2 these transitional basal progenitors differentiate into intestinal-like epithelium including goblet cells, thus reproducing Barrett’s metaplasia. A similar transitional columnar epithelium is present at the transitional zones of other mouse tissues, including the anorectal junction, and, importantly, at the gastro-oesophageal junction in the human gut. Acid reflux-induced oesophagitis and the multilayered epithelium (MLE) believed to be a precursor of BE are both characterized by the expansion of the transitional basal progenitor cells. Taken together our findings reveal the presence of a previously unidentified transitional zone in the epithelium of the upper gastrointestinal tract and provide evidence that the p63+ KRT7+ basal cells in this zone are the cell-of-origin for MLE and BE.

Synchrony between embryo competency and uterine receptivity is essential for successful implantation. Mice with ablation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) in the uterus (PR(Cre/+);COUP-TFII(flox/flox)) exhibit implantation defects and increased estrogen receptor (ER)alpha activity in the luminal epithelium, suggesting high ERalpha activity may disrupt the window of uterine receptivity. To determine whether increased ERalpha activity in the PR(Cre/+);COUP-TFII(flox/flox) uterus is the cause of defective implantation, we assessed whether inhibition of ERalpha activity could rescue the PR(Cre/+);COUP-TFII(flox/flox) uterine implantation defect. ICI 182,780 (ICI), a pure ERalpha antagonist, was administered to PR(Cre/+);COUP-TFII(flox/flox) mutant and COUP-TFII(flox/flox) control mice during the receptive period, and the number of implantation sites was examined. COUP-TFII(flox/flox) control mice treated with oil or ICI showed the normal number of implantation sites. As expected, no implantation sites were observed in PR(Cre/+);COUP-TFII(flox/flox) mutant mice treated with oil, consistent with previous observations. In contrast, implantation sites were greatly increased in ICI-treated PR(Cre/+);COUP-TFII(flox/flox) mutant mice, albeit at a reduced number in comparison with the control mice. ICI treatment was also able to restore the expression of Wnt4 and bone morphogenetic protein 2, important for endometrial decidualization in the PR(Cre/+);COUP-TFII(flox/flox) mutant mice. To confirm that the rescue of embryo attachment and decidualization is a consequence of a reduced ERalpha activity upon ICI treatment, we showed a reduction of the expression of ERalpha target genes in PR(Cre/+);COUP-TFII(flox/flox) mutant mice. Because COUP-TFII was also shown in our laboratory to be important for placentation during pregnancy, we asked whether ICI treatment could also rescue the placentation defect to allow full-term pregnancy in these mice. We found that whereas mice were born in COUP-TFII(flox/flox) control mice given ICI, no pups were born in the PR(Cre/+);COUP-TFII(flox/flox) mutant mice, suggesting that the increased ERalpha activity is not the reason for placentation defects. These results demonstrate that during the periimplantation period, COUP-TFII regulates embryo attachment and decidualization through controlling ERalpha activity. However, COUP-TFII expression is still required in the postimplantation period to facilitate placentation.