Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 210 pages. Includes Vita. Includes bibliographical references.

Little is known about higher order chromatin structures in the human brain and their function in transcription regulation. We employed chromosome conformation capture (3C) to analyze chromatin architecture within 700 Kb surrounding the transcription start site (TSS) of the NMDA receptor and schizophrenia susceptibility gene, GRIN2B, in human and mouse cerebral cortex. Remarkably, both species showed a higher interaction between the TSS and an intronic sequence, enriched for (KRAB) Krueppel associated Box domain binding sites and selectively targeted by the (H3K9) histone 3 lysine 9 specific methyltransferase ESET/SETDB1. Transgenic mice brain cortical nuclei over-expressing Setdb1 showed increased heterochromatin-protein 1 signal at the interacting regions coupled with decreased Grin2b expression. 3C further revealed three long distant chromatin loop interactions enriched with functional enhancer specific (H3K27Ac) histone 3 lysine 27 acetylation signal in GRIN2B expressing tissue (human cortical nuclei and Human Embryonic Kidney - HEK cells). Doxycycline-induced SETDB1 over-expression decreased 2 out of 3 loop interaction frequencies suggesting a possible SETDB1-mediated transcription repression. We also report a specific looping interaction between a region 50Kb upstream of the (GAD1) Glutamic Acid Decarboxylase – 1 gene TSS and the GAD1 TSS in human brain nuclei. GAD1 catalyzes the rate limiting step in (GABA) gamma amino-butyric acid synthesis and is quintessential for inhibitory signaling in the human brain. Clinical studies in schizophrenia brain samples reveal a decreased looping interaction frequency in correspondence with a decrease in gene expression. Our findings provide evidence for the existence of transcription relevant higher order chromatin structures in human brain.

Hormone-dependent cancers (HDCs), such as those of the prostate, ovary, breast and endometrium, share characteristics that indicate similar underlying mechanisms of carcinogenesis. Through steroid hormone signalling on "down-stream" target genes, the growth, development and progression of HDCs are regulated. One such family of target genes, highly expressed in HDCs and regulated by steroid hormones, are the tissue kallikreins (KLKs). The KLKs are a multigene family of serine proteases involved in physiological processes such as blood pressure regulation, inflammation, and tumour development and progression via the hydrolysis of specific substrates. Although the KLK gene family is clearly implicated in tumourigenesis, the precise roles played by these genes are largely unknown. Additionally, except for the androgen-responsive genes, KLK2 and KLK3, the mechanisms underlying their hormonal regulation in HDCs are yet to be identified. The initial focus of this thesis was to examine the regulation of the kallikreins, KLK1 and KLK4, by estradiol and progesterone in endometrial and breast cancer cell lines. From these studies, progesterone clearly regulated KLK4 expression in T47D cells and therefore, the focus of the remaining studies was to further examine this regulation at the transcriptional level. An overview of the results obtained is detailed below. Human K1 and hK4 protein levels were increased by 10 nmol/L estradiol benzoate, progesterone, or a combination of the two, over 48 hours in the endometrial cancer cell line, KLE. However, these same treatments resulted in no change in KLK1 gene or hK1 protein levels in the endometrial cancer cell lines, HEC1A or HEC1B (only hK1 analysed). Progesterone treatment (0-100 nmol/L) over 24 hours resulted in a clear increase in KLK4 mRNA at the 10 nmol/L dose in the breast cancer cell line, T47D. Additionally, treatment of T47D cells with 10 nmol/L progesterone over 0-48 hr, resulted in the rapid expression of the hK4 protein at 2 hr which was sustained for 24 hr. Further analysis of this latter progesterone regulation with the antiprogesterone, RU486, over 24 hours, resulted in an observable decrease in hK4 levels at 1 µmol/L RU486. Although the estrogen and progesterone regulation of the hK1 protein was not further analysed, the data obtained for hK4 regulation in T47D cell lines, supported the premise that this gene was progesterone-responsive. The rapid expression of hK4 protein by progesterone at two hours suggests that KLK4 transcription is directly coupled to progesterone regulation, perhaps through progesterone receptor (PR) binding to progesterone-responsive regions within the KLK4 promoter or far "up-stream" regions. Thus, the following further studies were performed. To test this hypothesis, the transcription initiation site (TIS) and 5' flanking regions of the KLK4 gene in T47D cells were interrogated. Primer extension and 5' RACE identified the TIS 78 bp 5' of the putative ATG site for translation as identified by Korkmaz et al. (2001). This KLK4 gene transcript consists of only four exons, and thus excludes the pre/pro signal peptide. Although a TATA-box is not present within -25 to -30 bp 5' of the identified TIS, a number of consensus binding motifs for Sp1 and estrogen receptor half-sites were identified. It is possible that the Sp1 sites are involved in the basal levels of transcription for this gene. Additionally, a putative progesterone response element (PRE) was identified in the far "up-stream" regions of the KLK4 gene. Basal levels of transcription were observed within the KLK4 proximal promoter region when coupled to a luciferase reporter gene and transfected into T47D cell lines. Additionally, the KLK4 proximal promoter region did not induce the luciferase reporter gene expression when progesterone was added to the system, however, estradiol was inhibitory for luciferase gene expression. This suggests that the proximal promoter region of the KLK4 gene could contain functional EREs but not PREs. In keeping with this hypothesis, some ER half-sites were identified, but PR sites were not obvious within this region. The identified PRE in the far "up-stream" region of the KLK4 gene assembled the progesterone receptor in vitro, and in vivo, as assessed by electromobility shift assays and chromatin immunoprecipitation assays (EMSAs and ChIPs), respectively. The binding of the PR to the KLK4 PRE was successfully competed out by a PR antibody and not by an androgen receptor antibody, and thus confirms the specificity of the KLK4 PRE-PR complex. Additionally, the PR was recruited and assembled onto and off the progesterone-responsive KLK4 region in a cyclic fashion. Thus, these data strongly suggest that the PR represents one of the core components of a transcription complex for the KLK4 gene, and presumably also contributes to the expression of this gene. Moreover, these data suggest a functional coordination between the PR and the KLK4 progesterone-responsive region in T47D cells, and thus, provide a model system to further study these events in vivo.