Enhancer-bound LDB1 regulates a corticotrope promoter-pausing repression program.

Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type.

Enhancer activation requires trans-recruitment of a mega transcription factor complex.

Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.

A protective strategy against hyperinflammatory responses requiring the nontranscriptional actions of GPS2.

The association between hyperinflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, nontranscriptional role of GPS2 as a guardian against hyperstimulation of the TNF-α-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNF-α target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the nontranscriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of Resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis.

Atbf1 is required for the Pit1 gene early activation.

Enhancers have been functionally described for >35 years, but the molecular principles underlying the integration of regulatory inputs to alternate gene enhancers used during mammalian organogenesis remain incompletely understood. Using a combination of in vivo enhancer mapping and proteomics approaches, we have established that two distant and distinct early enhancers, each requiring different transcription complexes, are required for full activation of the gene encoding the pituitary lineage determining factor, Pit1. A transcription factor belonging to the "giant, multiple-homeodomain and zinc finger family," Atbf1, serves as a novel pituitary regulator for one of the two required enhancers as shown by genetic and in vitro analysis.

Opposing LSD1 complexes function in developmental gene activation and repression programmes.

Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1--a component of the CoREST-CtBP co-repressor complex--is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

Homeodomain-mediated beta-catenin-dependent switching events dictate cell-lineage determination.

While the biological roles of canonical Wnt/beta-catenin signaling in development and disease are well documented, understanding the molecular logic underlying the functionally distinct nuclear transcriptional programs mediating the diverse functions of beta-catenin remains a major challenge. Here, we report an unexpected strategy for beta-catenin-dependent regulation of cell-lineage determination based on interactions between beta-catenin and a specific homeodomain factor, Prop1, rather than Lef/Tcfs. beta-catenin acts as a binary switch to simultaneously activate expression of the critical lineage-determining transcription factor, Pit1, and to repress the gene encoding the lineage-inhibiting transcription factor, Hesx1, acting via TLE/Reptin/HDAC1 corepressor complexes. The strategy of functionally distinct actions of a homeodomain factor in response to Wnt signaling is suggested to be prototypic of a widely used mechanism for generating diverse cell types from pluripotent precursor cells in response to common signaling pathways during organogenesis.

Critical roles of the p160 transcriptional coactivators p/CIP and SRC-1 in energy balance.

Several transcriptional coactivators have been implicated in modulating the transcriptional activities of nuclear hormone receptors in vitro. Potential roles of these cofactors in important physiological processes such as energy homeostasis remain unknown. We report here that a developmental arrest in interscapular brown fat and defective adaptive thermogenesis occur in mice lacking both the p160 family transcriptional coactivators SRC-1 and p/CIP due to a failure in induction of selective PPARgamma target genes involved in adipogenesis and mitochondrial uncoupling. In the absence of p/CIP and SRC-1, mice eat more food on both regular chow and a high-fat diet because of decreased blood leptin levels. However, the p/CIP(-/-)/SRC-1(-/-) mice are lean and resistant to high-fat-diet-induced obesity. They exhibit increased basal metabolic rates and heightened levels of physical activity. Therefore, p/CIP and SRC-1 play critical roles in energy balance by controlling both energy intake and energy expenditure.

Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis.

The precise mechanistic relationship between gene activation and repression events is a central question in mammalian organogenesis, as exemplified by the evolutionarily conserved sine oculis (Six), eyes absent (Eya) and dachshund (Dach) network of genetically interacting proteins. Here, we report that Six1 is required for the development of murine kidney, muscle and inner ear, and that it exhibits synergistic genetic interactions with Eya factors. We demonstrate that the Eya family has a protein phosphatase function, and that its enzymatic activity is required for regulating genes encoding growth control and signalling molecules, modulating precursor cell proliferation. The phosphatase function of Eya switches the function of Six1-Dach from repression to activation, causing transcriptional activation through recruitment of co-activators. The gene-specific recruitment of a co-activator with intrinsic phosphatase activity provides a molecular mechanism for activation of specific gene targets, including those regulating precursor cell proliferation and survival in mammalian organogenesis.