Forkhead box (Fox) family transcription factors are highly conserved and play essential roles in a wide range of cellular and developmental processes. This family was named after the ectopic head structures observed in mutants of the Drosophila gene forkhead (fkh). Since the discovery of fkh, hundreds of Fox genes have been identified in organisms ranging from yeasts to humans, making it one of the largest but least explored families of higher eukaryotic transcription factors. The NIH Undiagnosed Diseases Program (NIH UDP), a clinical site of the NIH Undiagnosed Diseases Network (UDN), identified a variant (p.M280L) in a single allele of the FOXR1 gene in an individual with severe neurological symptoms including postnatal microcephaly, progressive brain atrophy, and global developmental delay. The de novo missense variant in FOXR1 converts a highly conserved methionine residue at amino acid 280 to leucine and was predicted to contribute to the individual’s disease. The goal of this research is to investigate the biological role of FOXR1 and to determine how the M280L mutant leads to disease pathogenesis. At the protein level, the M280L mutant impaired FOXR1 expression and induced a nuclear aggregate phenotype when overexpressed in HEK 293T and COS7 cells due to protein misfolding and proteolysis. A FOXR1 C-terminal truncation mutant mimicked the M280L phenotype, suggesting that the C-terminal sequences of FOXR1 are important for FOXR1 protein stability. RNAseq and pathway analysis in HEK 293T cells indicated that FOXR1 acts as both transcriptional activator and repressor, playing central roles in heat shock response, chaperone cofactor-dependent protein refolding, and cellular response to stress. Indeed, FOXR1 expression is increased in HEK 293T in response to cellular stress, a process in which FOXRI directly controls HSPA6, HSPA1A and DHRS2 transcription. In contrast, the ability of the M280L mutant to respond to stress is compromised, in part due to impaired regulation of downstream target genes that are involved in the stress response pathway. Combined, these results suggest that FOXR1 plays a role in cellular stress and that impairment of these functions may contribute to the disease phenotypes seen in the individual with the FOXRI M280L variant.

Includes abstract. / Includes bibliographical references (leaves 155-181). / Researchers have demonstrated the ability to predict psychopathological states from human peripheral immune tissure transcriptional profiles, using microarrays. Although evidence in support of such an approach as a viable diagnostic avenue within psychiatric settings is accumulating, it remains to be demonstrated, in an animal model, that transcriptional changes in peripheral tissue targets are paralleled by specific gene expression changes in neural tissues.

Includes bibliographical references (leaves 78-88). / The unique skeletal structure of the bat forelimb and hindlimb provides a new and exciting model for the field of evolutionary developmental biology, which seeks to reveal the molecular mechanisms behind vertebrate limb diversity. The digits of the bat forelimb, excluding the thumb, are considerably elongated and webbed. The hindlimb digits are free of webbing and are of uniform length, lacking the asymmetrical patterning of the forelimb. In this study, gene expression analysis has revealed that changes in the spatial and temporal expression patterns of the anteriorposterior patterning signal, Sonic hedgehog (Shh), and its downstream target, Patched 1 (PtcJ), have contributed to the development of the unique bat limb. The embryonic development of Miniopterus natalensis (Miniopteridae) is described for the first time and the expression patterns of Shh and PtcJ in the developing limbs of this species are compared to those in Carollia perspicillata (Phyllostomidae) and the mouse. Early in bat limb development (stage 14), Shh expression in the ZPA appears to be anteriorly expanded when compared to the mouse. This observation is in line with the reported expansion of Fgf8 expression in the AER (Cretekos et al. 2007) and reveals that an enhancement of the Shh-Fgf positive feedback loop may be responsible for the initial posterior expansion of the bat forelimb. Later in development (stage 16) Shh and PtcJ acquire a novel domain of expression within the interdigital tissue of both the bat forelimb and hindlimb. These expression patterns parallel the reported up-regulation of Fgf8, Gremlin and Bmp2 in the interdigital tissue of C. perspicillata (Weatherbee et al. 2006) and support the hypothesis that the Shh-Fgfpositive feedback loop is re-initiated in the interdigital tissue of the bat limbs. The cell survival and proliferation signals provided by the Shh-Fgf signalling loop most likely contribute to the lengthening of the posterior forelimb digits, the survival of the tissue between the forelimb digits and the extension of digits 1 and 5 of the hindlimb to the same length of the remaining digits. The novel Shh and PtcJ expression patterns were observed in both M natalensis and C. perspicillata, supporting the monophyly of the chiropteran sub-order, Verspertilioniformes.

Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate of two different tyrosine kinases. First, c-Abl kinase phosphorylates YY1 at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies revealed that c-Abl mediated phosphorylation of YY1 regulated YY1’s transcriptional ability in vivo. Secondly, we show that YY1 is phosphorylated by non-receptor tyrosine kinase Src and this phosphorylation is mediated by the receptor tyrosine kinase c-Kit signaling pathway at tyrosine residue 251. Computational prediction using GPS 3.0 identified Src as a possible kinase that could target YY1 for tyrosine phosphorylation. The use of a highly sensitive phospho-specific antibody against phosphorylated Y251 in combination with non-radioactive in vitro kinase assay show that Src phosphorylates YY1 in vitro. Pharmacological inhibition of both c-Kit and Src kinase caused a great reduction in tyrosine phosphorylation of YY1 at Y251. The use of SCF ligand to stimulate c-Kit kinase show that YY1 may be a target of Src kinase phosphorylation under the c-Kit signaling cascade at Y251. Ongoing research includes the generation of phospho-mutations at tyrosine 251 using the CRISPR/Cas9 genome editing tool to uncover the biological significance of this phosphorylation. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1’s transcriptional activity, linking YY1 regulation with the c-Abl tyrosine kinase signaling pathways. We also link YY1 phosphorylation to the c-Kit receptor tyrosine kinase signaling pathway. Because errors in signaling result in cancer growth and other disease, understanding the dynamic cellular processes of YY1 phosphorylation by tyrosine kinases will lead to a better understanding of the signaling networks within the cell leading to more effective treatment for disease. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2017. / November 8, 2017. / c-Abl kinase, phosphorylaion, phospho-specific antibodies, transcription factor, tyrosine, YY1 / Includes bibliographical references. / Myra M. Hurt, Professor Directing Dissertation; Brian P. Chadwick, University Representative; Akash Gunjan, Committee Member; Cathy Levenson, Committee Member.

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The majority of the mammalian genome comprises non-coding regions, where numerous long non-coding RNAs (lncRNAs) are transcribed. Although it has been revealed that some lncRNAs serve as key regulators in essential biological processes, the majority of their functions remain unknown, notably in liver metabolism. Analysis of our recent genome-wide screen data reveals that Gm15441, a lncRNA antisense to a protein-coding gene thioredoxin-interacting protein (Txnip), is the most significantly upregulated lncRNA in the fasting mouse liver. Given that certain antisense lncRNAs have been reported to regulate the expression of their sense genes, the roles of Gm15441 in regulating Txnip expression were addressed in this work. Furthermore, the fact that Txnip is a prominent regulator of liver metabolism prompted us to investigate the functional role of the Gm15441-Txnip axis in liver metabolic homeostasis.We first investigated the response of Gm15441 and Txnip to in vivo metabolic challenges, such as fasting and refeeding, and in vitro metabolic stimuli, such as insulin and key metabolic transcription factors. Next, in mouse hepatocytes, we assessed the regulation of Txnip expression by Gm15441 and the underlying mechanism. Lastly, using an adenovirus-mediated liver-specific overexpression approach, we determined whether Gm15441 regulates Txnip expression in the mouse liver and modulates key aspects of liver metabolism, including glucose and lipid metabolism. We found that Gm15441 is most robustly upregulated in the mouse liver upon fasting. In addition, Gm15441 and Txnip expression levels were shown to respond similarly to various metabolic signals both in vivo and in vitro, although their functions were predicted to be opposed. Furthermore, knockdown of Gm15441 by siRNA showed a trend of increasing Txnip protein expression in hepatocytes, while overexpression of Gm15441 by adenoviral gene delivery dramatically reduced Txnip protein expression both in vitro and in vivo. Mechanistically, we showed that Gm15441 directly binds to Txnip at RNA levels and inhibits Txnip translation. Moreover, the 207-709 nucleotides of Gm15441, which is complementary to the Txnip 5’end, mediate the reduction of Txnip protein expression. Lastly, we found that liver-specific overexpression of Gm15441 by adenovirus regulates glucose and lipid metabolism by decreasing blood glucose and plasma triglyceride levels while increasing plasma ketone body levels. In conclusion, our study demonstrated that lncRNA Gm15441 is a potent translational inhibitor of Txnip and a critical regulator in the liver metabolic homeostasis. This study characterizes both gene modulation and biologic functional role of Gm15441 and reveals the therapeutic potential of lncRNA Gm15441 for the treatment of glucose and lipid metabolic disorders. / Biomedical Sciences

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