Regulation of gene expression by DNA-protein and protein -protein interactions
Abstract
Transcriptions factors include a class of regulatory proteins that bind DNA and control the transcription of protein-coding genes. NF-IL6, c-Fos, and c-Jun are examples of regulatory proteins that contain a bZIP domain for DNA binding and protein dimerization. In order to study protein-protein interactions, several c-Fos peptides were synthesized and tested for their ability to inhibit c-Jun homodimers. The design of this de novo strategy was based on reported studies of c-Fos and c-Jun. The results suggested that synthetic peptides formed heterodimers with c-Jun in a sequence-specific manner, probably through the leucine zipper region of c-Jun. In order to study DNA-protein interactions and to understand the principles of gene regulation in human cells, the control region of the human NF-IL6 gene was studied. The segments selected for analysis include module A (−426/+38), module E (−3327/−2977) and module F (−3647/−3555). These segments were tested in DNA-binding and transient expression assays. The results of DNA-binding experiments provided evidence for potential protein-binding elements that might regulate the expression of the NF-IL6 gene. Protein-binding sequences included a response element, a κB element, and sites that interacted with Sp1/Sp3, Sp1/Sp2/Sp3, EGR-1, USF-1/USF-2, CREB and NF-IL6. In addition, the experiments identified several potential composite elements at positions −247/−204, −3156/−3148, and −3606/−3598 of the gene. Functional assays examined several deletion mutants, made in different regions of the NF-IL6 proximal promoter. The results suggested complex modes of regulation involving the responsive element and sites interacting with κB, Sp1/Sp3, and EGR-1. Studies of potential distal regulatory regions identified an element responsive to LPS and PMA in module F. The transcription factor database in the laboratory was updated to study the relative distribution of transcription factor genes in human chromosomes. The updated database includes more than 750 Pol II regulatory genes. Density analysis identified clusters of transcription factor genes in human chromosomes. Clusters were found in chromosomes 2, 7, 12 and 17, known to contain the Hox genes. However, the analysis also identified clusters of transcription factor genes in chromosomes 5, 6 and 22.
Degree
Ph.D.
Advisors
Bina, Purdue University.
Subject Area
Biochemistry|Organic chemistry|Molecular biology
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