To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.

We describe the cloning, characterization, and tissue distribution of the two human peroxisome proliferator activated receptor isoforms hPPAR␥2 and hPPAR␥1. In cotransfection assays the two isoforms were activated to approximately the same extent by known PPAR␥ activators. Human PPAR␥ binds to DNA as a heterodimer with the retinoid X receptor (RXR). This heterodimer was activated by both RXR agonists and antagonists and the addition of PPAR␥ ligands with retinoids resulted in greater than additive activation. Such heterodimer-selective modulators may have a role in the treatment of PPAR␥/RXR-modulated diseases like diabetes. Northern blot analysis indicated the presence of PPAR␥ in skeletal muscle, and a sensitive RNase protection assay confirmed the presence of only PPAR␥1 in muscle that was not solely due to fat contamination. However, both PPAR␥1 and PPAR␥2 RNA were detected in fat, and the ratio of PPAR␥1 to PPAR␥2 RNA varied in different individuals. The presence of tissue-specific distribution of isoforms and the variable ratio of PPAR␥1 to PPAR␥2 raised the possibility that isoform expression may be modulated in disease states like non-insulin-dependent diabetes mellitus. Interestingly, a third protected band was detected with fat RNA indicating the possible existence of a third human PPAR␥ isoform.

Retinoid X receptor (RXR) plays a central role in the regulation of many intracellular receptor signalling pathways and can mediate ligand-dependent transcription, acting as a homodimer or as a heterodimer. Here we identify an antagonist towards RXR homodimers which also functions as an agonist when RXR is paired as a heterodimer to specific partners, including peroxisome proliferator-activated receptor and retinoic acid receptor. This dimer-selective ligand confers differential interactions on the transcription machinery: the antagonist promotes association with TAF110 (TATA-binding protein (TBP)-associated factor 110) and the co-repressor SMRT, but not with TBP, and these properties are distinct from pure RXR agonists. This unique class of RXR ligands will provide a means to control distinct target genes at the level of transcription and allow the development of retinoids with a new pharmacological action.

An important function of interleukin-1 (IL-1) is activation of the transcription factor NF-kappa B, which is signaled via the type I IL-1 receptor (IL-1RI). By receptor mutagenesis studies, we have identified a region of the cytoplasmic domain of IL-1RI that is required for both IL-1-mediated NF-kappa B activation and IL-1-dependent activation of a receptor-associated protein kinase activity we term IRAK. No IL-1RI mutants were found that can activate NF-kappa B in the absence of IRAK activity. Therefore, we propose that IRAK activation is a necessary step in the activation of NF-kappa B by IL-1.

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Promoter-and enhancer-binding factors appear to function by facilitating the transcription reaction as well as by counteracting chromatin-mediated repression (antirepression). We have examined the mechanism by which a hybrid activator, GAL4-VP16, is able to counteract histone HI-mediated repression by using both H1-DNA complexes and reconstituted HI-containing chromatin templates. The GAL4 DNA bind{ng domain alone was sufficient to disrupt local H1-DNA interactions, but a transcriptional activation region was additionally necessary for antirepression. GAL4--VP16-mediated antirepression required an auxiliary factor, denoted as a co-antirepressor, which was partially purified from Drosophila embryos. We have found that the co-antirepressor activity was sensitive to digestion with RNase A. Moreover, total RNA from Drosophila embryos could partially substitute for the co-antirepressor fraction, which indicated that the co-antirepressor may function as a histone acceptor ("histone sink"}. These findings suggest a model for gene activation in which sequence-specific transcription factors disrupt H1-DNA interactions at the promoter to facilitate transfer of HI to a histone acceptor, which then allows access of the basal transcription factors to the DNA template.[Key Words: Transcriptional regulation; histone H1; chromatin; RNA polymerase II; in vitro transcription] Received July 16, 1992; revised version accepted September 29, 1992.The proper control of gene expression is essential for the development, growth, and sustenance of eukaryotic organisms, yet the strategies and mechanisms by which genes are regulated remain to be clarified. An early step in the pathway leading to gene expression is initiation of transcription. Synthesis of mRNA is carried out by the RNA polymerase II transcriptional machinery, which comprises RNA polymerase II and several auxiliary factors that are commonly referred to as general factors (for recent reviews, see Saltzman and Weinmann 1989;Sawadogo and Sentenac 1990;Conaway and Conaway 1991; Zawel and Reinberg 1992). Transcription by the basal transcriptional apparatus is regulated by sequencespecific DNA-binding factors that interact with promoter and enhancer elements (for review, see Johnson and McKnight 1989;Mitchell and Tjian 1989), and it presently appears that many of these promoter-and enhancer-binding proteins may stimulate transcription by acting in conjunction with another class of factors that are referred to as coactivators, mediators, adaptors, or intermediary factors {for review, see Lewin 1990; Ptashne and Gann 1990; Pugh and Tjian 1992}. Transcriptional activity is also affected by chromatin structure {for review, see Weintraub 1985;Elgin 1988;Gross and Garrard 1988; van Holde 1989;Grunstein 1990;Wolffe 1990Wolffe , 1992 Felsenfeld 1992}; thus, it is important to consider the function of the general transcriptional machinery, the promoter-and enhancer-binding factors, and the coactivators with the chromatin template. 1991;To study the relationship between chromatin structure ...

This report describes the development of an orally active analgesic that resolves inflammation and neuropathic pain without the addictive potential of opioids. EC5026 acts on the cytochrome P450 branch of the arachidonate cascade to stabilize epoxides of polyunsaturated fatty acids (EpFA), which are natural mediators that reduce pain, resolve inflammation, and maintain normal blood pressure. EC5026 is a slow-tight binding transitionstate mimic that inhibits the soluble epoxide hydrolase (sEH) at picomolar concentrations. The sEH rapidly degrades EpFA; thus, inhibiting sEH increases EpFA in vivo and confers beneficial effects. This mechanism addresses disease states by shifting endoplasmic reticulum stress from promoting cellular senescence and inflammation toward cell survival and homeostasis. We describe the synthesis and optimization of EC5026 and its development through human Phase 1a trials with no drug-related adverse events. Additionally, we outline fundamental work leading to discovery of the analgesic and inflammationresolving CYP450 branch of the arachidonate cascade.

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