Molecular Mechanisms of Nuclear Hormone Receptor Transcriptional Synergy and Autoinduction.

Thyroid hormone (TH) and glucocorticoids (GC) play critical roles in the development and function of the central nervous system (CNS) by binding to their cognate nuclear hormone receptors (NRs), which function as ligand-activated transcription factors. In my dissertation, I studied the TH and GC-mediated regulation of Krüppel like factor 9 (Klf9/Basic Transcription Element Binding Protein 1; Bteb1), a member of the Sp1/KLF family of zinc finger transcription factors that bind GC rich genomic sequences, and plays an important role in neuronal development and plasticity. In prior work Klf9 was found to be a direct TH receptor (TR) target gene. I showed that Klf9 is also directly targeted by the GC receptor (GR), and that TH and GCs cause synergistic induction of Klf9. This synergistic regulation is phylogenetically ancient, and was likely present in the earliest tetrapods and has been evolutionarily conserved from frogs to mammals. I identified a genomic region in the 5’ flanking region of the Klf9 gene (the ‘Klf9 synergy module’) that contains TR/GR binding sites and confers synergistic gene regulation by TH and GC. The synergistic effect of TH and GC on Klf9 can be explained by a TR-dependent increase in the recruitment of the GR and enhanced association of stalled RNA polymerase II at the Klf9 synergy module, and an interaction between the synergy module and the Klf9 promoter by chromosomal looping. I also conducted a genome-wide microarray analysis, the first study to identify transcriptional targets that are coordinately regulated by TH and GC in the brain. Lastly, I demonstrated a role for KLF9 as an accessory transcription factor to support TH-dependent expression of TRβ (autoinduction), a process that is necessary for the progression of amphibian metamorphosis, and normal mammalian brain development. Given the synergistic regulation of the Klf9 gene by TR and GR, and its role in TRβ autoinduction, my findings support that KLF9 is an important intermediate that functions to integrate TH and GC by enhancing the cell sensitivity to hormonal signals. Taken together, my thesis broadens our understanding of the molecular mechanisms of NR cooperativity and autoinduction, with important implications for animal development.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/93977/1/piab_1.pd

Coordinated transcriptional regulation by thyroid hormone and glucocorticoid interaction in adult mouse hippocampus-derived neuronal cells.

The hippocampus is a well-known target of thyroid hormone (TH; e.g., 3,5,3'-triiodothyronine-T3) and glucocorticoid (GC; e.g., corticosterone-CORT) action. Despite evidence that TH and GC play critical roles in neural development and function, few studies have identified genes and patterns of gene regulation influenced by the interaction of these hormones at a genome-wide scale. In this study we investigated gene regulation by T3, CORT, and T3 + CORT in the mouse hippocampus-derived cell line HT-22. We treated cells with T3, CORT, or T3 + CORT for 4 hr before cell harvest and RNA isolation for microarray analysis. We identified 9 genes regulated by T3, 432 genes by CORT, and 412 genes by T3 + CORT. Among the 432 CORT-regulated genes, there were 203 genes that exhibited an altered CORT response in the presence of T3, suggesting that T3 plays a significant role in modulating CORT-regulated genes. We also found 80 genes synergistically induced, and 73 genes synergistically repressed by T3 + CORT treatment. We performed in silico analysis using publicly available mouse neuronal chromatin immunoprecipitation-sequencing datasets and identified a considerable number of synergistically regulated genes with TH receptor and GC receptor peaks mapping within 1 kb of chromatin marks indicative of hormone-responsive enhancer regions. Functional annotation clustering of synergistically regulated genes reveal the relevance of proteasomal-dependent degradation, neuroprotective effect of growth hormones, and neuroinflammatory responses as key pathways to how TH and GC may coordinately influence learning and memory. Taken together, our transcriptome data represents a promising exploratory dataset for further study of common molecular mechanisms behind synergistic TH and GC gene regulation, and identify specific genes and their role in processes mediated by cross-talk between the thyroid and stress axes in a mammalian hippocampal model system

AVR/NAVR deficiency lowers blood pressure and differentially affects urinary concentrating ability, cognition, and anxiety-like behavior in male and female mice

Arginine vasopressin (AVP) and angiotensin II (ANG II) are distinct peptide hormones involved in multiple organs modulating renal, cardiovascular, and brain functions. They achieve these functions via specific G protein-coupled receptors, respectively. The AVR/NAVR locus encodes two overlapping V2-type vasopressin isoreceptors: angiotensin-vasopressin receptor (AVR) responding to ANG II and AVP equivalently, and nonangiotensin vasopressin receptor (NAVR), which binds vasopressin exclusively. AVR and NAVR are expressed from a single gene by alternative promoter usage that is synergistically upregulated by testosterone and estrogen. This study tested the hypothesis that AVR/NAVR modulates urinary concentrating ability, blood pressure, and cognitive performance in vivo in a sex-specific manner. We developed a C57BL/6 inbred AVR/NAVR−/− knockout mouse that showed lower blood pressure in both male and female subjects and a urinary-concentrating defect restricted to male mice. We also detected sex-specific effects on cognitive and anxiety-like behaviors. AVR/NAVR−/− male mice exhibited impaired visuospatial and associative learning, while female mice showed improved performance in both type of cognition. AVR/NAVR deficiency produced an anxiolytic-like effect in female mice, while males were unaffected. Analysis of AVR- and NAVR-mediated phosphorylation/dephosphorylation of signaling proteins revealed activation/deactivation of known modulators of cognitive function. Our studies identify AVR/NAVR as key receptors involved in blood pressure regulation and sex-specific modulation of renal water homeostasis, cognitive function, and anxiety-like behavior. As such, the AVR/NAVR receptor system provides a molecular mechanism for sexually diergic traits and a putative common pathway for the emerging association of hypertension and cognitive decline and dementia