Retinoic acid, thyroid hormone, and vitamin D receptors preferentially activate target genes through response elements that consist of direct repeat arrangements of a core recognition motif of consensus sequence AGGTCA. We present evidence that the preference for direct repeat elements arises from two fundamental differences from steroid hormone receptors. First, retinoic acid, thyroid hormone, and vitamin D receptors are demonstrated to preferentially form heterodimers with the retinoid X receptors. These interactions are mediated by the carboxy-terminal dimerization interface, with heterodimer preference specified by actions of the DNA-binding domain. Second, the DNA-binding domains of heterodimeric receptors appear to be rotationally flexible with respect to the carboxy-terminal dimerization interface. Several independent lines of evidence suggest that, relative to the retinoid X and steroid hormone receptors, the DNA-binding domain of the thyroid hormone receptor is preferentially rotated by -180 ~ with respect to its carboxy-terminal dimerization interface. As a result, solution interactions between the carboxy-terminal dimerization interfaces of the retinoid X and thyroid hormone receptors are predicted to lead to the preferential alignment of their respective DNA-binding domains in a direct repeat configuration. This alignment would position the retinoid X receptor over the upstream recognition motif of direct repeat response elements. Differential orientations of the DNA-binding domain, which contribute to the polarity of heterodimer binding, are regulated by a short sequence (the A box) that is located between the conserved DNA-binding and carboxy-terminal dimerization domains.[Key Words: Nuclear receptor heterodimersl DNA-binding domain~ carboxy-terminal dimerization interface] Received April 9, 1993~ revised version accepted May 13, 1993.The thyroid hormone, retinoic acid, and vitamin D receptors [TR, RAR, and VDR, respectively} are members of the nuclear receptor superfamily of ligand-dependent transcription factors that interact with response elements in target genes and thereby control diverse aspects of development and homeostasis (Evans 1988~ Beato 1989~ Glass and Rosenfeld 1991}. A central problem in understanding the actions of these and related nuclear receptors is the elucidation of the molecular mechanisms by which target genes are recognized. Studies of the DNA-binding properties of the TR, RAR, and VDR SCorgesponding author. 6Present address:

Macrophages are key immune cells found in atherosclerotic plaques and critically shape atherosclerotic disease development. Targeting the functional repertoire of macrophages may hold novel approaches for future atherosclerosis management. Here, we describe a previously unrecognized role of the epigenomic enzyme Histone deacetylase 3 (Hdac3) in regulating the atherosclerotic phenotype of macrophages. Using conditional knockout mice, we found that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and thus induces a stable plaque phenotype. Also, macrophages presented a switch to anti-inflammatory wound healing characteristics and showed improved lipid handling. The pro-fibrotic phenotype was directly linked to epigenetic regulation of the Tgfb1 locus upon Hdac3 deletion, driving smooth muscle cells to increased collagen production. Moreover, in humans, HDAC3 was the sole Hdac upregulated in ruptured atherosclerotic lesions, Hdac3 associated with inflammatory macrophages, and HDAC3 expression inversely correlated with pro-fibrotic TGFB1 expression. Collectively, we show that targeting the macrophage epigenome can improve atherosclerosis outcome and we identify Hdac3 as a potential novel therapeutic target in cardiovascular disease.

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Recent studies indicate that heterodimeric nuclear receptors utilize several novel mechanisms for increasing the complexity of transcriptional responses to hormonal stimuli. By binding as heterodimers, these receptors can potentially respond to more than one activating ligand. Allosteric interactions between the ligand binding domains of RXR and its heterodimeric partners regulate the binding of RXR ligands, resulting in either selective or dual transcriptional responses. Regulation of the relative levels of expression of different heterodimeric partners that permit signaling through RXR is likely further to expand the patterns of transcriptional responses that can occur through a given response element. Heterodimeric nuclear receptors also bind to asymmetric response elements with specific polarities that result from the formation of cooperative interfaces between DNA binding domains. The DNA binding interface serves to determine the response element specificity of different heterodimers based on the spacing between half sites. The specific polarity of DNA binding has also been shown to provide a mechanism for regulating the transcriptional responses of retinoic acid receptors to activating ligands through the differential control of co-repressor interactions. The identification and characterization of co-activator and co-repressor molecules is likely to provide a very interesting next chapter to the mechanisms of steroid hormone action.

Thiazolidinediones (TZDs), known as peroxisome proliferator-activated receptor (PPAR) agonists, are used to treat type 2 diabetes. However, ∼5% of patients experience the treatment-limiting side effect of edema. Studies have implicated activation of the epithelial sodium channel (ENaC) as a cause of TZD-induced fluid retention, although there have been conflicting reports. The goal of this study was to resolve the role of PPARγ in control of ENaC isoforms in the kidney. Herein, we demonstrate in mice that rosiglitazone (RGZ), a PPARγ ligand, increases body weight and abdominal fat pad fluid content and reduces hematocrit. Seven days of RGZ decreases ENaCα and ENaCβ mRNA and ENaCγ protein expression in the kidney cortex, and acute treatment for 5 h with pioglitazone, another potent TZD, does not increase renal ENaC isoform mRNA or protein expression. Pioglitazone also decreases ENaCα and ENaCγ mRNA expression in a cortical collecting duct cell line. As no direct transcriptional studies had been conducted, we examined the PPARγ-dependent regulation of ENaC. Pioglitazone represses ENaCγ promoter activity, and this repression is partially relieved by inhibition of protein synthesis. Chromatin immunoprecipitation assays revealed that repression is associated with a decrease in histone H4K5 acetylation at the proximal ENaCγ promoter. In summary, TZDs do not increase ENaC mRNA expression in the kidney, and in fact repress the ENaCγ promoter via an indirect transcriptional mechanism.