Chapter 2. Aims & Objectives

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2 2.1. Statement of the problem: Earlier reports have shown ambiguous alteration of histone marks in response to DNA damage in asynchronized population of cells. These histone marks not only undergo dynamic alteration in context of DNA damage but are also known to alter during cell cycle in order to maintain phase specific chromatin states. This might lead to differential radio-sensitivity in different phases of cell cycle. Previous studies suggest that in response to DNA damage, cells in late S-phase are more radio-resistant whereas G2/M-phase cells are less radio-resistant compared to G1-phase cells. The different repair pathways are predominantly active during specific cell cycle stages. Like, homologous recombination (HR) is predominant in late S-phase and G2/M phase and non-homologous end-joining (NHEJ) is predominant in G1-phase. The specific histone marks might associate with repair pathways and distinct chromatin states in response to DNA damage in different phase of cell cycle Hypothesis We hypothesize that the histone marks that are induced at the site of DNA damage could be different and are associated with specific phase of the cell cycle and repair machinery. Therefore, the cell cycle phase specific histone marks may be the key determinants for phase specific differential response to ionizing radiation. The aim of the present study is to investigate cell cycle phase specific alteration of histone marks and chromatin structure in response to ionization irradiation and to delineate the proteins associated with histone modifications. 31

3 Accordingly, the objectives of the present research work are: 2.3. Objectives Whether any alterations in histone modifications/variants is associated with specific cell cycle stage that alter chromatin structure for DNA repair in response to double strand DNA damage? What are the interacting protein partners of altered histone(s) in response to double strand DNA damage? The following experiments were conducted on cultured human cells as a model system in order to address the objectives: 2.4. Whether any alterations in histone modifications/variants is associated with specific cell cycle stage that alter chromatin structure for DNA repair in response to double strand DNA damage? Time dependent cell cycle profiling after irradiation of G1 enriched cell lines (WRL68 and HepG2) by flow cytometry. Time dependent immunofluorescence staining of γh2ax in G1 enriched cell lines (WRL68 and HepG2) after irradiation in order to understand prime, repair and restoration phase of DNA damage response. Time dependent alteration of histone marks profiling in G1 enriched cell lines (WRL68 and HepG2) after irradiation by western blotting. Dual immunofluorescence staining of different histone marks after irradiation in order to understand co-localization with γh2ax (DNA damage mark). 32

4 Synchronization of cells in different phases of cell cycle in order to validate altered histone marks and its association with specific phase of cell cycle. Induction of different type of DNA damage in order to validate universal nature of altered histone marks in G1 phase of cell cycle. Enrichment of cells in G1 phase of different tissue origin in order to demonstrate altered histone marks in response to DNA damage is independent of tissue origin and universal in nature. MNase sensitivity assay to demonstrate global changes in chromatin organization at the nucleosomal level in response to IR induced DNA damage in G1 phase of cell cycle. Mononucleosomal immunoprecipitation experiment to demonstrate alteration of DNA damage responsive histone marks on same γh2ax bearing mononucleosomes in response to IR induced DNA damage in G1 phase of cell cycle. 2.5 What are the interacting protein partners of altered histone(s) in response to double strand DNA damage? Protein expression profile of downstream effectors of MAP Kinase pathway by western blotting in order to establish its association with altered histone marks after irradiation. In-silico docking studies to demonstrate interaction between effector enzymes with its respective altered histone marks. Biochemical fractionation studies to demonstrate localization of effector enzymes to altered chromatin structure in response to DNA damage in G1 phase of cell cycle. 33

5 Mononucleosomal immunoprecipitation to demonstrate direct interaction of effector enzymes with γh2ax bearing mononucleosomes in response to DNA damage in G1 phase of cell cycle. Inhibitor-based approaches for mechanistic studies of alteration of histone marks in response to DNA damage. Functional studies such as colony formation assay for survival, cell viability by trypan blue staining and DNA damage repair by comet assay to demonstrate significance of effector enzymes and altered histone marks in DNA damage response. 34