Author

Paromita Deb

ORCID Identifier(s)

0000-0002-9903-2592

Graduation Semester and Year

2017

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Subhrangsu Mandal

Abstract

Endocrine disrupting chemicals (EDCs) are classes of environmental contaminants whose exposure even at very low concentrations interfere with normal endocrine signaling and induce many adverse health effects including increased risk of birth defects, diabetes, obesity, abnormal reproduction, and cancer. EDC exposure alters the epigenetic programming of cells resulting in altered gene expression and regulation and cell signaling. These EDCs include bisphenol-A (BPA), diethylstilbesterol (DES), polychlorinated biphenyl (PCB), phthalates, phytoestrogens, methoxychlor, fungicides, insecticide, herbicide, and some heavy metals. As a part of my work, I have investigated epigenetic impacts of EDCs on HOX (homeobox containing genes) genes, which are critical players in embryonic development. They determine segmental identity along the anterio-posterior (AP) body axis. HOX genes are also expressed in adults for functional differentiation. Notably, deregulation of HOX gene expression contributes to severe human diseases including cancer. The knowledge on the regulatory mechanisms that drive HOX expression is limited. Studies demonstrate that HOX gene expressions are influenced by variety of hormones that include estradiol, progesterone, testosterone, retinoic acid, vitamin D and others. By regulating HOX genes, hormonal signals utilize a conserved mechanism that allows generation of structural and functional diversity in both developing and adult tissues. In my research, I have investigated the mechanism of endocrine regulation (by estradiol) and disruption (by EDCs) of a particular HOX gene, HOXB9, in vitro and in vivo. Notably, HOXB9 is a homeobox-containing gene that plays key roles in mammary gland development and is associated with breast and other types of cancer. Here, we demonstrate that HOXB9 expression is transcriptionally regulated by estradiol (E2), in vitro and in vivo. We also demonstrate that endocrine disrupting chemical BPA induces HOXB9 expression in cultured human breast cancer cells (MCF7) as well as in vivo in the mammary glands of ovariectomized (OVX) rats. Luciferase assay showed that estrogen-response-elements (EREs) at the HOXB9 promoter are induced by BPA. Estrogen-receptors (ERs) and ER-coregulators such as mixed lineage leukemia 3 (MLL3) histone methylase, histone acetylases, CBP/p300, bind to the HOXB9 promoter EREs in the presence of BPA, modify chromatin (histone methylation and acetylation) and lead to gene activation. In summary, our results demonstrate that BPA exposure alters the epigenetic programming of the HOXB9 promoter leading to its endocrine disruption in vitro and in vivo. Hypoxia signaling plays a critical role in tumor growth, angiogenesis, metastatic cancer, and aging. Under hypoxic condition, hypoxia-inducible factors (HIFs) are stabilized and they coordinate the process of hypoxia-induced gene expression and cell signaling pathways and leads to increased tumor cell proliferation, angiogenesis and growth. Recent studies indicate that non-coding RNAs which are closely associated with cancer are abnormally expressed under hypoxia. Here, we have investigated the transcriptional regulation of a cancer associated long non-coding RNA (lncRNA), homeobox transcript antisense RNA (HOTAIR) under hypoxic conditions. Our studies demonstrate that HOTAIR expression is upregulated under hypoxia in colon cancer and several other types of cancer cells. HOTAIR transcription is regulated by HIF1α which binds to the hypoxia response elements (HRE) present in the HOTAIR promoter under hypoxia. HIF1α knockdown results in decreased HOTAIR expression under hypoxia. Along with HIF1α, histone acetyltransferase, p300, histone methyl-transferase, MLL1, and RNA polymerase II are enriched at the HOTAIR promoter in a hypoxia dependent manner. The levels of H3K4-trimethylation and histone acetylation are also enriched at the HOTAIR promoter under hypoxic conditions. Overall, our studies demonstrate that HOTAIR expression is induced under hypoxic environment via coordination of HIFs and other transcription activators which may contribute to its roles in tumorigenesis. Long noncoding RNAs are abundant in the mammalian transcriptome and many of them are specifically expressed in the brain. HOTAIR, a long intergenic noncoding RNA (lincRNA), is known to be overexpressed in several diseases. However, the role of HOTAIR in neurons/neurological disorders remains unclear. Neurotransmitters and neurotrophic factors are critical to neuronal signaling, growth and neuroprotection. Brain derived nerotrophic factor (BDNF) is a critical neurotrophic factor that plays key roles in neuronal growth, maintenance and protection and abnormal levels of BDNF in brain causes neurological disorders. Here we have investigated the mechanism of BDNF expression, especially, mediated by lncRNA HOTAIR. Our studies have demonstrated that HOTAIR is a key regulator of BDNF. Knockdown of HOTAIR resulted in induction of BDNF expression in hippocampal (HT22) cells. Furthermore, enzymatic inhibition of a HOTAIR-interacting partner, EZH2, also resulted in BDNF induction. Chromatin immunoprecipitation analysis demonstrated that levels of EZH2 (HOTAIR-interacting histone trimethylase) and LSD1 (HOTAIR-interacting histone demethylase) were reduced at the BDNF promoter under EZH2-inhibition condition. The level of repressive histone modification marks such as H3K27-trimethylation (which is introduced by EZH2) is decreased at the BDNF promoter. Taken together, these observations indicate that HOTAIR along with with interacting partners such as EZH2 are involved in repression of BDNF expression. Beyond lncRNA, estrogen is also known to modulate neural plasticity, cognition and neuroprotection. Indeed, our studies demonstrate that estradiol (E2) treatment induces the expression of BDNF mRNA and protein levels in HT22 cells in vitro. Estrogen receptor ERα/ERβ and ER-coregulators (CBP/p300) and MLL3 were enriched at the BDNF promoter in the presence of E2. Additionally, we showed that E2 induced-BDNF expression is mediated by the displacement of silencing factors, EZH2 and LSD1 at BDNF promoter and subsequent recruitment of active transcription machinery. These results reveal the function and mechanisms of lncRNA, HOTAIR to enforce silent chromatin state at BDNF promoter which in turn is rescued upon treatment with positive regulators such as E2.

Keywords

Homeobox genes, 17β-estradiol, Estrogen receptor, Estrogen response element, Activation protein-1, Specific-protein 1, Nuclear receptor co-repressor, Histone deacetylase, Histone acetylase, CREB-binding protein, Polycomb repressive complex 2, Brain-derived neurotrophic factor, Oxytocin, Bisphenol-A, Chromatin immunoprecipitation, Diethyl pyrocarbonate, Diethylstilbestrol, Dulbecco’s modified eagle’s medium, Dithiothreitol, Ethylenediaminetetraacetic acid, Fetal bovine serum, HOX antisense intergenic RNA, Long non-coding RNA, Mixed lineage leukemia, Moloney murine leukemia virus, Nuclear receptor, Phosphate buffered saline, Phenylmethanesulfonylfluoride, Progesterone receptor, Retinoic acid receptor, Hypoxia inducible factors, Von-Hippel Lindau tumor suppressor gene, Hypoxia response element, Factor inhibiting HIF-1, Proline hydroxylase, Reverse transcriptase polymerase chain reaction, Quantitative polymerase chain reaction, Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Steroid receptor coactivator-1, Suppressor of Zeste 12 homolog, Retinoblastoma associated protein 46/48, Embryonic ectoderm development, Enhancer of Zeste homolog 2, Lysine-specific demethylase-1, Ovariectomized, Small nuclear RNAs, Small nucleolar RNAs, Transcription start-site-associated RNAs, Promoter-associated small RNAs, Promoter upstream transcripts, Anterio-posterior axis, Retinoid X receptor, Silencing mediator of retinoic acid, Vitamin D receptor, Vitamin D response element, Nerve growth factor, Neurotrophin 3/4, Fibroblast growth factor, Transforming growth factor, Vascular endothelial growth factors, Erythropoietin, Ciliary neurotrophic factor, Leukemia inhibitory factor, Piwi-interacting RNA, Enhancer RNA, Urothelial carcinoma associated 1, Estrogen-related receptors, Antisense RNA, RE1-silencing transcription factor, Extracellular signal-regulated kinase, Polycyclic aromatic hydrocarbons, Polychlorinated biphenyl, Polybrominated biphenyls, Hypothalamus pituitary gonadal, Homeobox transcript antisense RNA

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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