All ETDs from UAB

Advisory Committee Chair

Jay M McDonald

Advisory Committee Members

Sumant Chugh

Robert Hardy

Selvarangan Ponnazhagan

Thomas Ryan

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Heme oxygenase-1 (HO-1) is a critical enzyme catalyzing the degradation of heme and generating carbon monoxide, iron, and biliverdin. In addition to heme degradation, HO-1 expression is known to protect against various cellular insults and disease states including acute kidney injury, atherosclerosis, vascular restenosis, and others. Human HO-1 gene expression is tightly regulated at the level of transcription. The main goal of this study is to explore the transcriptional regulation of the human HO-1 gene in renal epithelial cells. Chromosome Conformation Capture (3C) demonstrates that multiple regulatory regions within the HO-1 promoter and enhancer regions are physically interacting with each other by forming chromatin loops and this looping is required for the initiation of the human HO-1 gene transcription. ChIP-Loop assay reveals that these regions share several common transcription factors such as Sp1, USF-1, and JunB. Sp1 binds to the intronic enhancer which interacts with the -4.5kb promoter region by chromatin looping. Inhibition of Sp1 by siRNA abolishes this interaction and reduces HO-1 transcription. To study human HO-1 transcription in vivo, a HO-1 bacterial artificial chromosome (BAC) transgenic mouse was generated by integrating a 87 kb BAC DNA (a portion of the human chromosome 22 including HO-1 gene and its regulatory regions) into the mouse genome. Global overexpression of HO-1 mRNA and protein in tissues of HO-1 BAC transgenic mice were detected. Moreover, using this HO-1 BAC transgenic mouse, a "humanized" HO-1 BAC transgenic mouse (hHO-1 BAC) was generated by crossing HO-1 BAC mice with HO-1 knockout (HO-1-/-) mice. In addition to the overexpression of human-specific HO-1 mRNA and protein expression, the human HO-1 gene in hHO-1 BAC mice rescued the abnormal phenotype observed in the HO-1-/- mice such as increased incidence of abortion and embryonic lethality, heightened sensitivity to acute kidney injury, iron overload, anemia, and splenomegaly. These studies provide not only functional insights for the regulation of the human HO-1 gene but also the molecular architecture of the human chromosome containing the HO-1 gene and promoter in renal epithelial cells.

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