| Post-translational modifications of histones (e.g. acetylation, methylation, phosphorylation and ubiquitination) are crucial for the regulation of chromatin structure and function; these modifications are thought to be interdependent, playing important roles in regulating transcriptional activity of DNA. Evidence has been provided that histones are also modified by the covalent binding of biotin; biotinylation of histones is mediated by holocarboxylase synthetase (HCS) and biotinidase (BTD). It has been proposed that biotinylated histones play a role in cell proliferation, gene silencing, and cellular response to DNA damage. Identification of biotinylation sites in histones is the first step in deciphering the cross-talk between biotinylation and other modifications of histones that regulate gene expression, and in understanding physiological roles of biotinylated histones. My Dissertation research addressed two major aims. In part one, I sought to identify amino acid residues that are biotinylated in histone H4, and to characterize the cross-talk between biotinylation and other known modifications in histone H4. In this study I used synthetic peptides and polyclonal antibodies to identify two biotinylation sites in human histones: lysine-8 and lysine-12 in histone H4. I also observed that acetylation and biotinylation compete for the same lysine residue, and that acetylation and methylation of histones may affect biotinylation of neighboring lysines. Subsequent studies in our laboratory have identified other biotinylation sites in histone H2A and H3, using the techniques developed and presented here.; Part two of my Dissertation work focused on identifying the genotype and phenotype associated with HCS and BTD deficiency using the fruit fly Drosophila melanogaster as a model. Knock down of HCS and BTD expression was associated with decreased histone biotinylation. HCS deficiency altered the expression of more than 500 genes; this was associated with decreased lifespan and tolerance to heat. The lifespan of HCS- and BTD-deficient flies decreased compared with wild-type flies. Likewise, HCS-deficient flies exhibited a considerably decreased tolerance to heat; effects in BTD-deficient flies were minor. This study was the first to provide evidence for an effect of HCS deficiency on gene function and phenotype in Drosophila . |
