Date of Award

2009

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Philippe Georgel

Second Advisor

Eric Blough

Third Advisor

Elaine Hardman

Fourth Advisor

Richard Niles

Fifth Advisor

Vincent Sollars

Abstract

The eukaryotic genome is packaged into chromosomes that are made up of a highly organized and heavily regulated structure called chromatin. The proteins involved in the compaction of DNA into this condensed state are mostly understood at the level of the structure of the nucleosome. The higher order arrangement of chromatin and how it effects gene regulation is only partially understood and characterized. The compaction of nucleosomal arrays into 30-nm and higher structures are partially the responsibility of architectural, or structural, chromatin associated proteins. The following dissertation analyzes the individual chromatin contributions of two well studied architectural proteins, the yeast silencing protein Silent Information Regulator 3 (Sir3) and the human transcriptional regulator methyl CpG binding protein 2 (MeCP2). Silencing in yeast is the responsibility of the SIR family of proteins. Classically, the Sir3 protein has been characterized as associating with chromatin through the hypo-acetylated N-termini of the core histones H3 and H4. The Sir3 protein has recently been found to contain a DNA-binding element, my studies characterized Sir3-nucleic acid interactions and showed that Sir3 can bind to chromatin independently of histone N-termini. In contrast, the MeCP2 protein has classically been characterized as a methylated DNA dependent transcriptional repressor, but recent genome-wide analysis reveals MeCP2 distribution can occur on promoters of active genes. Recent in vitro work with MeCP2 and nucleosomal arrays showed a highly ordered, compacted chromatin structure even in the absence of DNA methylation. MeCP2 is of particular biological interest due to the observed link with the neurodevelopmental iii disorder Rett Syndrome (RTT). My studies demonstrated that MeCP2 can bind in vitro to the Ntermini of core histones H2A, H3, and H4. Additionally, the removal of these tails impacted MeCP2-chromatin interactions, and resulted in a reduced level of nucleosomal array condensation. Importantly, the two RTT mutants analyzed here, R133C and R168X, exhibited differential binding to histone N-termini. These results add to the understanding of chromatin organization and arrangement by demonstrating and characterizing additional chromatin contacts for these two chromatin associated proteins.

Subject(s)

Chromatin.

DNA-binding proteins.