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Description
Schizophrenia affects 1.1% of the population worldwide. Schizophrenia is a complex, multifactorial disorder. Stress can trigger psychotic episodes and exacerbate schizophrenic symptoms. For humans, one gene implicated in stress and schizophrenia in humans is the early growth response 3 (EGR3). Patients with genomic variations in EGR3 have reduced levels of EGR3 in the prefrontal brain region compared with healthy patients. Schizophrenic patients also have less serotonin 2A receptor (5HT2AR), which is coded by the gene Htr2a, in their prefrontal cortex. Mice that are Egr3-deficient also have decreased levels of 5HT2AR, suggesting that Egr3 may be involved in the regulation of 5HT2AR. The purpose of the experiment is to determine if EGR3 binds to the Htr2a gene promoter region by using a Chromatin immunoprecipitation (ChIP) assay. We will use ECS to increase EGR3 expression. Previously we have identified two upstream sites of interest where EGR3 potentially binds to the Htr2a gene, one which is distal and one proximal to the transcription start site. After ECS, increased binding is seen in the Htr2a distal region with EGR3 via the ChIP assay. Increased binding was not observed at either of the promoter sites; however, the t-test comparing the distal site of the ECS and the No ECS groups to have a p-value of 0.056, suggesting that increasing the number of animals (n=7) could possibly give a more accurate representation to test our hypothesis. However, the experiment still suggests increased expression and that EGR3 may bind to the distal site of Htr2a. Keywords: stress, environment, genetics, schizophrenia, EGR3, chromatin immunoprecipitation
ContributorsMishra, Abhinav (Author) / Buetow, Kenneth (Thesis director) / Gallitano, Amelia (Committee member) / Zhao, Xiuli (Committee member) / Barrett, The Honors College (Contributor) / School of Politics and Global Studies (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Schizophrenia is considered a multifactorial disorder with complex genetic variants in response to environmental stimuli. However, the specific genetic contribution to schizophrenia risk is largely unknown. The transcription factor early growth response gene 3 (EGR3) can be activated rapidly after stimuli and thus may translate environmental stimuli into gene changes that influence schizophrenia risk. However, the downstream genes that may be regulated by EGR3 are not clear. While the 5-Hydroxytryptamine receptor 2A (5HT2AR) - encoding gene Htr2a has been implicated in the etiology of schizophrenia, the mechanisms by which Htr2a influences susceptibility to this illness are poorly understood. We previously found that in addition to schizophrenia-like abnormalities, Egr3 -/- mice have approximately 70% deduction of 5HT2AR level in the prefrontal cortex, which underlines their resistant to the sedating effect of clozapine. These findings indicate that the two schizophrenia candidate genes are in the same biological pathway that integrates multiple components resulting in schizophrenia. This dissertation is aimed to identify the mechanisms by which Egr3 regulates the expression of Htr2a in response to environmental stimuli like stress.
To determine if Egr3 alters Htr2a transcription under stress, I examined messenger ribonucleic acid (mRNA) levels of these two genes in wildtype (WT) and Egr3 -/- mice after 6hrs of sleep deprivation (SD). I found both genes are increased in WT mice after SD compared with controls. In addition, Egr3 is required for Htr2a induction because SD fails to induce Htr2a expression in Egr3 -/- mice. Next, I performed chromatin immunoprecipitation (ChIP) to determine if EGR3 binds to Htr2a promoter in vivo. I found a significant increase of EGR3 binding to Htr2a distal promoter 2hrs after seizure. To determine the functionality of this binding, I co-transfected the CMV- EGR3 vector or CMV- vector alone with the Htr2a distal promoter reporter clone. I found overexpression of EGR3 activates the Htr2a distal promoter-driven luciferase gene. Although the ChIP assay shows no direct binding of EGR3 to Htr2a proximal promoter, I found EGR3 overexpression activates Htr2a proximal promoter-driven luciferase gene. These findings suggest that EGR3 regulates Htr2a probably through both direct and indirect ways.
To determine if Egr3 alters Htr2a transcription under stress, I examined messenger ribonucleic acid (mRNA) levels of these two genes in wildtype (WT) and Egr3 -/- mice after 6hrs of sleep deprivation (SD). I found both genes are increased in WT mice after SD compared with controls. In addition, Egr3 is required for Htr2a induction because SD fails to induce Htr2a expression in Egr3 -/- mice. Next, I performed chromatin immunoprecipitation (ChIP) to determine if EGR3 binds to Htr2a promoter in vivo. I found a significant increase of EGR3 binding to Htr2a distal promoter 2hrs after seizure. To determine the functionality of this binding, I co-transfected the CMV- EGR3 vector or CMV- vector alone with the Htr2a distal promoter reporter clone. I found overexpression of EGR3 activates the Htr2a distal promoter-driven luciferase gene. Although the ChIP assay shows no direct binding of EGR3 to Htr2a proximal promoter, I found EGR3 overexpression activates Htr2a proximal promoter-driven luciferase gene. These findings suggest that EGR3 regulates Htr2a probably through both direct and indirect ways.
ContributorsZhao, Xiuli (Author) / Gallitano, Amelia (Thesis advisor) / Van Keuren-Jensen, Kendall (Committee member) / Lifshitz, Jonathan (Committee member) / Neisewander, Janet (Committee member) / Arizona State University (Publisher)
Created2017