Stressing Out: Dynamics Of Chromatin Insulator Body Formation In Eukaryotes Under Osmotic Stress

Higher order genome organization and the role it plays in governing cell dynamics and protein expression has become a widely studied field. Chromatin insulators are important to this organization in their ability to form long range contacts between distant regions on the genome. During times of osmotic stress, insulator proteins leave their binding sites on the DNA to form insulator bodies in the nucleus. This phenomenon relieves the DNA of its structure and is rapidly reversible. Using a variety of immunofluorescent staining methods, this work looked to further characterize this process while attempting to identify molecular mechanisms that mediate chromatin response to osmotic stress. Our laboratory has shown that insulator proteins co-localize with [gamma] H2Av, an important signaling molecule whose main function is marking sites of DNA damage. Here, we further confirmed that the histone variant H2Av is necessary for stability of the Drosophila gypsy insulator and that this function may be mediated by Ataxia telangiectasia (ATM) and Ataxia telangiectasia and Rad3 related (ATR). We find that phosphorylation of H2Av controls the insulator response to osmotic stress. In addition, we ask whether insulators also have a role in the osmotic response in human cells, and show how human cells react to osmotic stress in a way similar to that of Drosophila. Overall this work builds on previous attempts to characterize insulator bodies and the role of ATM and ATR in genome architecture and function.