Deoxyribonucleic acid (DNA) and histone proteins are the building blocks of nucleosomes, repeating units of chromatin. Both DNA and histone proteins are methylated, and these methylated molecules are bound by proteins with specialized reader domains. The E3 ubiquitin ligases UHRF1 and UHRF2 are two such proteins with domains that recognize bothmethylated DNA and methylated histones. UHRF1 participates in the maintenance of DNA methylation through cell division, yet the relationship between each domain activity remains unclear. Using UHRF1 and UHRF2 as model systems to study molecular mechanisms of multivalent chromatin interactions and allosteric regulation of enzymatic activity, we sought to understand how interactions with methylated histones, methylated DNA, and methylation events on other proteins influenced the enzymatic activities and DNA methylation regulatory functions of these homologous chromatin-associated proteins. We identified key components of both chromatin and the UHRF proteins that regulate E3 ligase activity. Further, we determined that recognition of methylated lysine by UHRF1, both on histone and non-histone proteins, was dispensable for DNA methylation maintenance. Throughout our studies, we made advances in the use of peptide microarray technologies to study chromatin binding protein domains and antibodies directed toward histone proteins. Finally, as a progression to the study of protein interactions with histones and DNA, we developed a streamlined protocol for chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to evaluate the genomic locations of chromatin features and interactors. The result was a rapid protocol that was quantitative and free of “spike-in” normalizers and was a step toward the physical understanding of a peak in ChIP-seq.
|Commitee:||Bedford, Mark T.|
|School:||Van Andel Research Institute|
|School Location:||United States -- Michigan|
|Source:||DAI-B 82/8(E), Dissertation Abstracts International|
|Subjects:||Biochemistry, Genetics, Molecular biology|
|Keywords:||E3 ubiquitin ligases, UHRF1 , UHRF2|
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