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Title:Regulatory dynamics of transcription factors TBX18 and WT1 in mouse prostate development
Author(s):Negi, Soumya
Director of Research:Stubbs, Lisa J
Doctoral Committee Chair(s):Stubbs, Lisa J
Doctoral Committee Member(s):Henry, Jonathan J; Li, Xin; Bolton, Eric C
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):T-box transcription factor Prostate development Mesenchymal differentiation Topologically associating domain Lineage tracing
Abstract:The T-box transcription factor TBX18 is essential to mesenchymal cell differentiation in several tissues and Tbx18 loss-of-function results in dramatic organ malformations and perinatal lethality. Although the expression and function of Tbx18 has been analyzed in several tissues, the homozygous mutants die at birth, and the long-term effects of Tbx18 in cell development and differentiation have not been identified. Using a long-lived translocation mutant, 12Gso - a hypomorphic allele of Tbx18 caused due to disruption of the gene’s regulatory structure - we were able to circumvent the perinatal lethality and investigate adult phenotypes of Tbx18 deficiency. Upon studying the histopathology of 12Gso/Tbx18- mutant adults, unexpectedly severe defects were seen in the prostate. The prostate became an ideal system to study Tbx18’s role in postnatal development due to its non-essential nature, thus giving us the ability to create conditional mutants of Tbx18 in this tissue that survive to adulthood. Our studies demonstrated that Tbx18 is essential to the development of periductal smooth muscle stromal cells in prostate, particularly in the anterior lobe, with a clear impact on prostate health in adult mice. Prostate mesenchymal cells that fail to express Tbx18 do not condense normally into smooth muscle cells of the periductal prostatic stroma; these periductal stromal cells in mutant prostate assume a hypertrophic, myofibroblastic state and the adjacent epithelium becomes grossly disorganized. To identify molecular events preceding the onset of this pathology, we compared gene expression in the urogenital sinus (UGS), from which the prostate develops, in Tbx18-null and wild type littermates at two embryonic stages. Genes that regulate cell proliferation, smooth muscle differentiation, prostate epithelium development, and inflammatory response were significantly dysregulated in the mutant urogenital sinus around the time that Tbx18 is first expressed in the wild type UGS, suggesting a direct role in regulating those genes. Our previous data indicated that the dynamic expression of Tbx18 is controlled by an expansive regulatory architecture, extending far upstream and downstream of the gene. We coupled circularized chromatin conformation capture (4C) and ATAC-seq from embryonic day 18.5 (E18.5) mouse urogenital sinus (UGS), with the goal of identifying elements that interact with the Tbx18 promoter in developing prostate. The data revealed dozens of active chromatin elements distributed throughout a 1.5 million base pair topologically associating domain (TAD). To identify cell types contributing to this chromatin signal, we used lineage tracing methods with a Tbx18:Cre “knock-in” allele; these data show clearly that Tbx18-expressing precursors differentiate into wide array of cell types in multiple tissue compartments, most of which had not been previously reported. We also used a 209 kb Cre-expressing Tbx18 transgene (Tbx18:BAC-iCre), to partition enhancers for specific precursor types into two rough spatial domains. RNA-seq studies of the developing UGS revealed expression of the zinc-finger transcription factor Wt1, a gene previously unreported to be expressed in the prostate and a known interaction partner of Tbx18. Furthermore, Wt1 was significantly dysregulated in Tbx18-/- mutant UGS indicating a relationship between the two genes in this tissue. After establishing high expression of Wt1 in the developing UGS and tracing the cell lineages arising from Wt1-expressing precursor cells, we obtained a conditional knockout mutation of the Wt1 gene and tested the effects of Wt1 loss of function in prostate development. Crossing the conditional mutants with animals carrying a prostate-specific Cre allele, we identified a severe over-proliferative phenotype in the prostate of the conditional Wt1 knockouts. To identify transcriptional targets of this TF, we performed WT1 chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) in UGS chromatin using a tested WT1 antibody. The data revealed a role for Wt1 upstream of the TGFβ/BMP pathway during prostate development, a signaling pathway known to inhibit over-proliferation of epithelial cells. Whereas WT1 has been known for some time to be dysregulated in human prostate cancer, its function in the normal prostate has not previously been described. This study has thus revealed a new role for the very well-studied WT1 protein, as a transcription factor with a key function in prostate development, and one with clear relevance to a potential link to prostate cancer in adults. This thesis uses a combination of traditional cell biology approaches along with functional genomics strategies to study the expression and function of the transcription factors, Tbx18 and Wt1, in prostate development for the first time. Using mouse genetics, we generate conditional mutants to identify histo-pathologies associated with the loss-of-function of these genes in the prostate. RNA-seq and ChIP-seq are also used to identify molecular targets and pathways downstream to these TFs. Finally, ATAC-seq and 4C are used to map the extended regulatory network that controls expression of Tbx18 in the particular cell fates.
Issue Date:2019-04-14
Type:Thesis
URI:http://hdl.handle.net/2142/105184
Rights Information:Copyright 2019 Soumya Negi
Date Available in IDEALS:2019-08-23
Date Deposited:2019-05


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