Biological Systems Engineering, Department of


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A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Agricultural and Biological Systems Engineering, Under the Supervision of Professors Angela K. Pannier and Janos Zempleni. Lincoln, Nebraska: December, 2010
Copyright 2010 Dipika Singh


Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. The objective of this project was to determine genomic binding sites of holocarboxylase synthetase (HCS) in the human chromatin of breast cell lines using an antibody independent technology. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to Dam for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation sensitive digestion, cloning, and sequencing. Fifteen novel HCS binding sites were identified and included sequences within genes coding for inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, myosin heavy chain 9 non-muscle, cadherins 8 and 11, prolactin, leptin precursor, schwannomin 1, apoptosis inhibitor, netrin-G1 ligand, neuron navigator, SRY-box4, purinergic receptor, and CUB and sushi domains. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.