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

The Escherichia coli GTPase CgtA(E) Is Involved in Late Steps of Large Ribosome Assembly

The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA(E), in late steps of large ribosomal subunit biogenesis. CgtA(E) belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA(E) cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtA(E) mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA(E) is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA(E). We also show that purified CgtA(E) associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA(E) cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants

Association of <i>ATP1A1</i> and <i>Dear</i> Single-Nucleotide Polymorphism Haplotypes With Essential Hypertension

Essential hypertension remains a major risk factor for cardiovascular and cerebrovascular diseases. As a complex multifactorial disease, elucidation of susceptibility loci remains elusive. ATP1A1 and Dear are candidate genes for 2 closely linked rat chromosome-2 blood pressure quantitative trait loci. Because corresponding human syntenic regions are on different chromosomes, investigation of ATP1A1 (chromosome [chr]-1p21) and Dear (chr-4q31.3) facilitates genetic analyses of each blood pressure quantitative trait locus in human hypertension. Here we report the association of human ATP1A1 ( P <0.000005) and Dear ( P <0.03) with hypertension in a relatively isolated, case/control hypertension cohort from northern Sardinia by single-nucleotide polymorphism haplotype analysis. Sex-specific haplotype analyses detected stronger association of both loci with hypertension in males than in females. Haplotype trend-regression analyses support ATP1A1 and Dear as independent susceptibility loci and reveal haplotype-specific association with hypertension and normotension, thus delineating haplotype-specific subsets of hypertension. Although investigation in other cohorts needs to be performed to determine genetic effects in other populations, haplotype subtyping already allows systematic stratification of susceptibility and, hence, clinical heterogeneity, a prerequisite for unraveling the polygenic etiology and polygene–environment interactions in essential hypertension. As hypertension susceptibility genes, coexpression of ATP1A1 and Dear in both renal tubular cells and vascular endothelium suggest a cellular pathogenic scaffold for polygenic mechanisms of hypertension, as well as the hypothesis that ATP1A1 and/or Dear could contribute to the known renal and vascular endothelial dysfunction associated with essential (polygenic) hypertension

CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is associated with resistance to androgen deprivation therapy, and an increase in the population of neuroendocrine (NE) differentiated cells. It is hypothesized that NE differentiated cells secrete neuropeptides that support androgen-independent tumor growth and induce aggressiveness of adjacent proliferating tumor cells through a paracrine mechanism. The cytochrome b561 (CYB561) gene, which codes for a secretory vesicle transmembrane protein, is constitutively expressed in NE cells and highly expressed in CRPC. CYB561 is involved in the α-amidation-dependent activation of neuropeptides, and contributes to regulating iron metabolism which is often dysregulated in cancer. These findings led us to hypothesize that CYB561 may be a key player in the NE differentiation process that drives the progression and maintenance of the highly aggressive NE phenotype in CRPC. In our study, we found that CYB561 expression is upregulated in metastatic and NE prostate cancer (NEPC) tumors and cell lines compared to normal prostate epithelia, and that its expression is independent of androgen regulation. Knockdown of CYB561 in androgen-deprived LNCaP cells dampened NE differentiation potential and transdifferentiation-induced increase in iron levels. In NEPC PC-3 cells, depletion of CYB561 reduced the secretion of growth-promoting factors, lowered intracellular ferrous iron concentration, and mitigated the highly aggressive nature of these cells in complementary assays for cancer hallmarks. These findings demonstrate the role of CYB561 in facilitating transdifferentiation and maintenance of NE phenotype in CRPC through its involvement in neuropeptide biosynthesis and iron metabolism pathways

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

Sex-specific hippocampus-dependent cognitive deficits and increased neuronal autophagy in DEspR haploinsufficiency in mice

Aside from abnormal angiogenesis, dual endothelin-1/VEGF signal peptide-activated receptor deficiency (DEspR−/−) results in aberrant neuroepithelium and neural tube differentiation, thus elucidating DEspR's role in neurogenesis. With the emerging importance of neurogenesis in adulthood, we tested the hypothesis that nonembryonic-lethal DEspR haploinsufficiency (DEspR+/−) perturbs neuronal homeostasis, thereby facilitating aging-associated neurodegeneration. Here we show that, in male mice only, DEspR-haploinsufficiency impaired hippocampus-dependent visuospatial and associative learning and induced noninflammatory spongiform changes, neuronal vacuolation, and loss in the hippocampus, cerebral cortex, and subcortical regions, consistent with autophagic cell death. In contrast, DEspR+/− females exhibited better cognitive performance than wild-type females and showed absence of neuropathological changes. Signaling pathway analysis revealed DEspR-mediated phosphorylation of activators of autophagy inhibitor mammalian target of rapamycin (mTOR) and dephosphorylation of known autophagy inducers. Altogether, the data demonstrate DEspR-mediated diametrical, sex-specific modulation of cognitive performance and autophagy, highlight cerebral neuronal vulnerability to autophagic dysregulation, and causally link DEspR haploinsufficiency with increased neuronal autophagy, spongiosis, and cognitive decline in mice

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