Analysis of Post-translational Modifications of Histones and Myelin Basic Protein: Crosstalk and Beyond.

Abstract

The four small, evolutionarily conserved histone proteins, H2A, H2B, H3 and H4, (core histones) play complex roles in eukaryotes, including gene transcription and DNA replication. Perhaps their most remarkable features are the numerous epigenetic marks [post-translational modifications (PTMs)] made by histone modifying enzymes. The combinatorial permutation of these chemical modifications constitutes the ‘histone code’ controling their roles in transcription, replication, and other functions. Initial efforts were directed towards the PTMs of myelin basic protein (described below), with the bulk of the effort directed towards analysis of histone PTMs. The histone goals were: 1) determine how histone PTMs are affected when the epigenetic system has been perturbed; 2) define the coordination of histone PTMs to control biological outcomes in the cell. I developed a new proteomic strategy to quantify histone PTMs in Tetrahymena thermophila using 15N metabolic labelling. I confirmed that a novel putative histone methyl transferase (HMT), Txr1p, preferentially effects monomethylation of H3K27. I also elucidated its relationship to Ezl2p, another HMT. I expanded this method to other core histones and identified several PTMs that exhibit cross-talk with methylation of H3K27. I also performed quantitative analysis of histone PTMs across 15 epigenetic features (growing, conjugating, or starving cells from WT and 7 mutants) to reveal crosstalk patterns in four histones. Multivariate analysis of histone PTM data revealed functionally related subgroups which significantly expands our understanding of the ‘histone code’ hypothesis and provides clues to its robustness. I also proposed a statistical model reflecting the relationships between the observed histone PTMs and the hidden features in these studies. I performed a detailed cross-species analysis of the myelin basic protein (MBP) PTMs from bovine and rattlesnake, revealing differences in MBP modification patterns between mammals and reptiles. This study extended our knowledge about relationships between MBP PTMs and demyelinating diseases. These two studies focused on the key hypothesis that proteins possess a ‘protein barcode’ enbedded in their large number of modifications to modulate their functions in response to physiological conditions.