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|Title:||From Technology to Chromatin Biology: Contemporary Mass Spectrometry of Histones and Chromatin Associated Proteins|
|Doctoral Committee Chair(s):||Kelleher, Neil L.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Interest in chromatin biology has increased dramatically in the past decade due to discoveries of its involvement in important cellular activities including epigenetics, development, gene activity, etc. Changes in chromatin at the molecular level have also been linked to human disease such as cancer and are highly important in determining cell fate. Post-translational modifications (PTMs) such as acetylation, methylation, and phosphorylation, are one of the mechanisms by which histones (the protein component of chromatin) signal changes in very dynamic nuclear networks. Here new types of mass spectrometry (MS) are used to decipher histone PTMs as they occur in combination, to generate a global view of all the modification networks throughout the genome, an initial step towards understanding the role they play human cells.
Developments in technology have revolutionized our view of how post-translational switches can effect protein function and mass spectrometry has certainly helped propel the chromatin field in new directions. Unlike other MS based techniques, top down mass spectrometry, preserves all modified histone forms given that the sample in analyzed at the intact protein level. This methodology also allows for characterization of highly similar protein variants which are members of the same family. Protein forms expressed by human cells (H2B) and different rat tissues (H2A and H2B) were determined by top down MS. Using electron capture dissociation (ECD) for MS/MS of H2B isoforms, direct evidence for the expression of eight unmodified H2B forms and monoacetylated H2B.A was obtained from asynchronous HeLa cells. Surprisingly, the expression profile of H2B forms did not change in HeLa cells arrested in mitosis by colchicine or during mid-S, mid-G2, G2/M, and mid-G1 phases of the cell cycle. H2A and H2B variants did not show expression dynamics in different subsections of rat brain or different rat tissues.
We have also developed a top down-like approach (middle down) which analyzes large peptides to characterize PTM profiles on histone H3. Interestingly, we were able to conclude that methylation and acetylation levels on histone H3 are highly effected by perturbations of DNA methylation. In the past years we have expanded the applications of top down and middle down MS to other chromatin-associated proteins like HMGA1. In this study we were able to link PTM dynamics (phosphorylation of Ser 43) on HMGA1a to DNA repair and the early events in apoptosis.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
|Date Available in IDEALS:||2014-12-17|