Co-regulation of nuclear respiratory factor-1 by NFkappaB and CREB links LPS-induced inflammation to mitochondrial biogenesis.

The nuclear respiratory factor-1 (NRF1) gene is activated by lipopolysaccharide (LPS), which might reflect TLR4-mediated mitigation of cellular inflammatory damage via initiation of mitochondrial biogenesis. To test this hypothesis, we examined NRF1 promoter regulation by NFκB, and identified interspecies-conserved κB-responsive promoter and intronic elements in the NRF1 locus. In mice, activation of Nrf1 and its downstream target, Tfam, by Escherichia coli was contingent on NFκB, and in LPS-treated hepatocytes, NFκB served as an NRF1 enhancer element in conjunction with NFκB promoter binding. Unexpectedly, optimal NRF1 promoter activity after LPS also required binding by the energy-state-dependent transcription factor CREB. EMSA and ChIP assays confirmed p65 and CREB binding to the NRF1 promoter and p65 binding to intron 1. Functionality for both transcription factors was validated by gene-knockdown studies. LPS regulation of NRF1 led to mtDNA-encoded gene expression and expansion of mtDNA copy number. In cells expressing plasmid constructs containing the NRF-1 promoter and GFP, LPS-dependent reporter activity was abolished by cis-acting κB-element mutations, and nuclear accumulation of NFκB and CREB demonstrated dependence on mitochondrial H(2)O(2). These findings indicate that TLR4-dependent NFκB and CREB activation co-regulate the NRF1 promoter with NFκB intronic enhancement and redox-regulated nuclear translocation, leading to downstream target-gene expression, and identify NRF-1 as an early-phase component of the host antibacterial defenses.

Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation.

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.

Impact of gene variants on sex-specific regulation of human Scavenger receptor class B type 1 (SR-BI) expression in liver and association with lipid levels in a population-based study.

BACKGROUND: Several studies have noted that genetic variants of SCARB1, a lipoprotein receptor involved in reverse cholesterol transport, are associated with serum lipid levels in a sex-dependent fashion. However, the mechanism underlying this gene by sex interaction has not been explored. METHODS: We utilized both epidemiological and molecular methods to study how estrogen and gene variants interact to influence SCARB1 expression and lipid levels. Interaction between 35 SCARB1 haplotype-tagged polymorphisms and endogenous estradiol levels was assessed in 498 postmenopausal Caucasian women from the population-based Rancho Bernardo Study. We further examined associated variants with overall and SCARB1 splice variant (SR-BI and SR-BII) expression in 91 human liver tissues using quantitative real-time PCR. RESULTS: Several variants on a haplotype block spanning intron 11 to intron 12 of SCARB1 showed significant gene by estradiol interaction affecting serum lipid levels, the strongest for rs838895 with HDL-cholesterol (p=9.2x10(-4)) and triglycerides (p=1.3x10(-3)) and the triglyceride:HDL cholesterol ratio (p=2.7x10(-4)). These same variants were associated with expression of the SR-BI isoform in a sex-specific fashion, with the strongest association found among liver tissue from 52 young women<45 years old (p=0.002). CONCLUSIONS: Estrogen and SCARB1 genotype may act synergistically to regulate expression of SCARB1 isoforms and impact serum levels of HDL cholesterol and triglycerides. This work highlights the importance of considering sex-dependent effects of gene variants on serum lipid levels.

The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.

A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.

Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX.

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

The Function and Regulation of Photobodies in Phytochrome Signaling

Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant Arabidopsis thaliana, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies.

Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.

Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (PBG), and one in which it cannot (NGB). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the NGB line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in NGB regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.

In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); hmr mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak hmr allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, SON OF HEMERA (SOH). The soh mutations rescue all of the phenotypes associated with the weak hmr allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null soh alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.

In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, SON OF HEMERA, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.

Genetic variants in the TEP1 gene are associated with prostate cancer risk and recurrence.

BACKGROUND: Telomere-related genes play an important role in carcinogenesis and progression of prostate cancer (PCa). It is not fully understood whether genetic variations in telomere-related genes are associated with development and progression in PCa patients. METHODS: Six potentially functional single-nucleotide polymorphisms (SNPs) of three key telomere-related genes were evaluated in 1015 PCa cases and 1052 cancer-free controls, to test their associations with risk of PCa. Among 426 PCa patients who underwent radical prostatectomy (RP), the prognostic significance of the studied SNPs on biochemical recurrence (BCR) was also assessed using the Kaplan-Meier analysis and Cox proportional hazards regression model. The relative telomere lengths (RTLs) were measured in peripheral blood leukocytes using real-time PCR in the RP patients. RESULTS: TEP1 rs1760904 AG/AA genotypes were significantly associated with a decreased risk of PCa (odds ratio (OR): 0.77, 95% confidence interval (CI): 0.64-0.93, P=0.005) compared with the GG genotype. By using median RTL as a cutoff level, RP patients with TEP1 rs1760904 AG/AA genotypes tended to have a longer RTL than those with the GG genotype (OR: 1.55, 95% CI: 1.04-2.30, P=0.031). A significant interaction between TEP1 rs1713418 and age in modifying PCa risk was observed (P=0.005). After adjustment for clinicopathologic risk factors, the presence of heterozygotes or rare homozygotes of TEP1 rs1760904 and TNKS2 rs1539042 were associated with BCR in the RP cohorts (hazard ratio: 0.53, 95% CI: 0.36-0.79, P=0.002 and hazard ratio: 1.67, 95% CI: 1.07-2.48, P=0.017, respectively). CONCLUSIONS: These data suggest that genetic variations in the TEP1 gene may be biomarkers for risk of PCa and BCR after RP.

Integrated pathway and epistasis analysis reveals interactive effect of genetic variants at TERF1 and AFAP1L2 loci on melanoma risk.

Genome-wide association studies (GWASs) have characterized 13 loci associated with melanoma, which only account for a small part of melanoma risk. To identify new genes with too small an effect to be detected individually but which collectively influence melanoma risk and/or show interactive effects, we used a two-step analysis strategy including pathway analysis of genome-wide SNP data, in a first step, and epistasis analysis within significant pathways, in a second step. Pathway analysis, using the gene-set enrichment analysis (GSEA) approach and the gene ontology (GO) database, was applied to the outcomes of MELARISK (3,976 subjects) and MDACC (2,827 subjects) GWASs. Cross-gene SNP-SNP interaction analysis within melanoma-associated GOs was performed using the INTERSNP software. Five GO categories were significantly enriched in genes associated with melanoma (false discovery rate ≤ 5% in both studies): response to light stimulus, regulation of mitotic cell cycle, induction of programmed cell death, cytokine activity and oxidative phosphorylation. Epistasis analysis, within each of the five significant GOs, showed significant evidence for interaction for one SNP pair at TERF1 and AFAP1L2 loci (pmeta-int  = 2.0 × 10(-7) , which met both the pathway and overall multiple-testing corrected thresholds that are equal to 9.8 × 10(-7) and 2.0 × 10(-7) , respectively) and suggestive evidence for another pair involving correlated SNPs at the same loci (pmeta-int  = 3.6 × 10(-6) ). This interaction has important biological relevance given the key role of TERF1 in telomere biology and the reported physical interaction between TERF1 and AFAP1L2 proteins. This finding brings a novel piece of evidence for the emerging role of telomere dysfunction into melanoma development.

A Quantitative Analysis of Growth and Size Regulation in Manduca sexta: The Physiological Basis of Variation in Size and Age at Metamorphosis.

Body size and development time are important life history traits because they are often highly correlated with fitness. Although the developmental mechanisms that control growth have been well studied, the mechanisms that control how a species-characteristic body size is achieved remain poorly understood. In insects adult body size is determined by the number of larval molts, the size increment at each molt, and the mechanism that determines during which instar larval growth will stop. Adult insects do not grow, so the size at which a larva stops growing determines adult body size. Here we develop a quantitative understanding of the kinetics of growth throughout larval life of Manduca sexta, under different conditions of nutrition and temperature, and for genetic strains with different adult body sizes. We show that the generally accepted view that the size increment at each molt is constant (Dyar's Rule) is systematically violated: there is actually a progressive increase in the size increment from instar to instar that is independent of temperature. In addition, the mass-specific growth rate declines throughout the growth phase in a temperature-dependent manner. We show that growth within an instar follows a truncated Gompertz trajectory. The critical weight, which determines when in an instar a molt will occur, and the threshold size, which determines which instar is the last, are different in genetic strains with different adult body sizes. Under nutrient and temperature stress Manduca has a variable number of larval instars and we show that this is due to the fact that more molts at smaller increments are taken before threshold size is reached. We test whether the new insight into the kinetics of growth and size determination are sufficient to explain body size and development time through a mathematical model that incorporates our quantitative findings.

Dissecting the Functional Impacts of Non-Coding Genetic Variation

A large proportion of the variation in traits between individuals can be attributed to variation in the nucleotide sequence of the genome. The most commonly studied traits in human genetics are related to disease and disease susceptibility. Although scientists have identified genetic causes for over 4,000 monogenic diseases, the underlying mechanisms of many highly prevalent multifactorial inheritance disorders such as diabetes, obesity, and cardiovascular disease remain largely unknown. Identifying genetic mechanisms for complex traits has been challenging because most of the variants are located outside of protein-coding regions, and determining the effects of such non-coding variants remains difficult. In this dissertation, I evaluate the hypothesis that such non-coding variants contribute to human traits and diseases by altering the regulation of genes rather than the sequence of those genes. I will specifically focus on studies to determine the functional impacts of genetic variation associated with two related complex traits: gestational hyperglycemia and fetal adiposity. At the genomic locus associated with maternal hyperglycemia, we found that genetic variation in regulatory elements altered the expression of the HKDC1 gene. Furthermore, we demonstrated that HKDC1 phosphorylates glucose in vitro and in vivo, thus demonstrating that HKDC1 is a fifth human hexokinase gene. At the fetal-adiposity associated locus, we identified variants that likely alter VEPH1 expression in preadipocytes during differentiation. To make such studies of regulatory variation high-throughput and routine, we developed POP-STARR, a novel high throughput reporter assay that can empirically measure the effects of regulatory variants directly from patient DNA. By combining targeted genome capture technologies with STARR-seq, we assayed thousands of haplotypes from 760 individuals in a single experiment. We subsequently used POP-STARR to identify three key features of regulatory variants: that regulatory variants typically have weak effects on gene expression; that the effects of regulatory variants are often coordinated with respect to disease-risk, suggesting a general mechanism by which the weak effects can together have phenotypic impact; and that nucleotide transversions have larger impacts on enhancer activity than transitions. Together, the findings presented here demonstrate successful strategies for determining the regulatory mechanisms underlying genetic associations with human traits and diseases, and value of doing so for driving novel biological discovery.

Posttranscriptional Regulation of Embryonic Neurogenesis by the Exon Junction Complex

The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.

Quantifying Eukaryotic Gene Regulation in Hormone Response and Disease.

Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.

Temporal Regulation of LMP1 and Apoptosis Resistance After Primary EBV Infection

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that establishes a lifelong latent infection in over ninety percent of all adult humans worldwide. While typically benign, EBV has been causally associated with a number of human malignancies in the settings of immune suppression, genetic, and/or environmental factors. While a highly successful pathogen based on prevalence, the ability of the virus to immortalize human B cells (a stage of infection thought to be critical for the establishment of latency) is quite poor. We hypothesize that the interactions between the virus and the human host early after infection are ultimately important for the outcome of viral latency establishment. To answer this question we broadly profiled primary human B cells at both early and late times after EBV infection to assay both host mRNA expression and the host-driven response to apoptotic stimuli. We found that EBV infection induces host gene expression signatures early after infection that are functionally distinct from the gene expression program late after infection. These studies also led to the novel discovery that viral gene expression is controlled differently early after infection, including the delayed expression of a viral protein that is critical for the establishment of latency. Furthermore, we have also shown that EBV can use a single viral protein to alter and repress host apoptotic sensitivity in the face of an anti-viral apoptotic response.