Elucidation of the Molecular Mechanisms Underlying Estrogen-Mediated Estrogen Receptor Activation.

摘要

In this study, we have focused on defining the mechanisms mediating gene expression in response to steroid hormones (predominantly 17beta-estradiol) as a model system for other non-steroid transcription systems that can be exploited to define general principals governing steroid responsiveness upon target genes. In particular, we have demonstrated that DNA sequence constraints define the functionally active steroid nuclear receptor (sNR) gene regulatory elements in the genome, and this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides, named nuclear receptor functional enhancers (NRFEs). At NRFEs, the chromatin binding of steroid nuclear receptors is not only correlated with active eRNA production, but also RNAPII occupancy as well as hormone-dependent coregulator and transcription factor (TF) recruitment. Moreover, steroid nuclear receptors with mutated DNA binding domains (DBD), were shown to still interact with chromatin, yet lack hormone-dependent transcriptional activity, highlighting the fact that steroid nuclear receptors can interact with chromatin in a transcriptionally inactive state (i.e., the majority of sNR chromatin interacting events identified in ChIP studies are not linked directly to transcriptional events) ( Chapter 2). We further demonstrate that the palindromic architecture of the regulatory element is the underlying mechanism that governs chromatin interaction by steroid nuclear receptors, and the non-functional chromatin interacting sites (non-NRFEs) observed in ChIP-seq studies are subject to the same rules and constraints as NRFEs. Thus, NRFE vs. non-NRFE binding is dictated at the individual nucleotide level, and the residence time (or strength of binding) is determined by the number and locations of variants within the consensus element. The basis of these rules and DNA constraints follow specific algebraic relationships. These findings quantitatively define how steroid nuclear receptors select the 'appropriate' regulatory targets out of a very large number of highly similar sequences in the genome, thus eliciting a specific cellular response (Chapter 3).;Estradiol is a potent mitogen in the mouse uterus, a well-characterized tissue used to study the underlying mechanisms of estrogen-mediated transcriptional regulation. Previously shown, estradiol-mediated transcriptional regulation in the mouse uterus is biphasic and can be divided into initial (early) phase and subsequent (late) phase transcriptional events. In this study, we demonstrate that late phase estradiol-mediated transcription requires the early phase transcripts and low-affinity estrogen receptor alpha (ERalpha) ligands cannot sustain late phase hormone-mediated transcriptional events. In addition, the interaction of ERalpha with chromatin is (1) immediate, (2) does not change locations after initial contact, (3) is retained the longest at NRFEs, and (4) is depleted prior to the late phase transcriptional events. Collectively, this indicates that estradiol-mediated late phase transcripts are regulated secondary to early induced transcripts (i.e., the early induced transcripts activate other transcription factors which are responsible for producing the late phase transcripts). Furthermore, the AF2 coactivator surface of ERalpha is not required for hormone-dependent ERalpha recruitment to NRFEs, estrogen-independent basal transcription requires ERalpha binding at NRFEs and the growth factor insulin-like growth factor 1 (IGF-1) activates ERalpha by recruitment of ERalpha to NRFEs (Chapter 4).;Our understanding of the physiology and transcriptional regulation of steroid hormones was significantly advanced following the generation of mutant mouse models possessing disruptions (knockouts) of the steroid nuclear receptor genes. In this study, we identified the molecular defects caused by a homozygous missense mutation in ERalpha identified in an 18 year-old woman with complete estrogen insensitivity syndrome; a clinical presentation similar to those of ERalpha knockout (alphaERKO) female mice. From these studies, we identified a potential therapeutic, Diethylstilbestrol (DES), for treating this estrogen insensitivity condition. Treatment of this patient with DES is underway and we remain involved as collaborators in this clinical study. Our studies also characterize the molecular defects caused by a different homozygous mutation in ERalpha identified in two sisters and a brother that likewise exhibit complete estrogen insensitivity (Chapter 5). (Abstract shortened by ProQuest.).