Date of Award

2007

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Environmental Science

First Advisor

Dr. Ahmed AI-Marzouqi

Second Advisor

Dr. Mahmoud Taleb AI-Ali

Third Advisor

Dr. Salman Ashraf

Abstract

Packaging of DNA into the condensed structure of chromatin presents a barrier to many cellular processes that require DNA access such as transcription and replication. This problem is solved, in part, by the action of various complexes that modify the chromatin structure so that it becomes more accessible and, therefore, a more suitable platform for these processes. ATP-dependent chromatin remodeling complexes and histone acetyltransferases are examples of many chromatin modifying complexes that work to alleviate chromatin-mediated repression. Histone H1 is an important component of chromatin which serves in both stabilizing and folding of chromatin into a higher order structure. Many studies have demonstrated the importance of histone H1 in gene regulation. Furthermore, histone H1 has been suggested to affect the functions of modifying proteins. In this study, we have used pull-down assay to test the effects of histone H1 on the binding of two chromatin modifying complexes (SWI/SNF and SAGA) to in vitro assembled unmodified and acetylated nucleosomal arrays. Gel shift assay was also performed to check for the effect of histone H1 on the binding of these complexes to mononucleosomes. Furthermore, we have tested the effects of histone H1 on the remolding activity of SWI/SNF using restriction enzyme accessibility assay. Our results show reduced binding of both SWI/SNF and SAGA complexes to both unmodified and acetylated nucleosomal array templates in the presence of histone H1. However, the histone H1-dependent inhibition of binding was specific to SWI/SNF when unmodified mononucleosomes were used. Furthermore, histone H1 was found to decrease the activity of the SWI/SNF complex.

Additionally, we investigated the effects of benzo[a]pyrene [B(a)P] on two cell lines, WRL-68 and HepG2 cells in an attempt to find whether chromatin modification is a possible pathway in the carcinogenesis of this compound. Benzo[a]pyrene is a chemical carcinogen that belongs to the polycyclic aromatic hydrocarbons, and is known to induce DNA damage by forming DNA adducts. We tested both cells lines with different concentrations of benzo[a]pyrene and for different durations. Cell viability and apoptosis were checked by cell cytotoxicity assay and flow cytometery, respectively followed by studies on the expression patterns of three chromatin remodeling proteins (BRG1, Gen5 and BAF155). Results show toxic effects of benzo[a]pyrene on both cells lines with increasing concentration and duration, while no changes in the expression of either BRG1, Gen5, and BAF155 was observed. This suggests that the carcinogenicity of benzo[a]pyrene doesn’t affect the expression pattern of at least these three proteins. However, our results don’t exclude chromatin modification as a possible pathway in the carcinogenesis of this compound. The expression of other chromatin modifying proteins need to be checked as well as the effects of B[a]P on the activity of these proteins before we can completely rule out a link between B[a]P effects on cells viability and chromatin

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