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DNA double-strand break (DSB) is considered as one of the most deleterious events for a cell because it poses the danger of genomic instability to a cell. An unproperly repaired cell can give rise to tumor if the cell goes through clonal expansion with the mutation from the unrepair damage. Chromatin structure, which can be controlled by epigenetic marks, is important for recognition and reparation of the DSB damaged. The DNA of DSB damaged must be in the “open” state for the repair factor to access and repair. Moreover, the DSB damage is also involved in the initiation of the upstream of Ataxia-telangiectasia mutated (ATM) signaling that follow after the presence of DSB. After DSB occurs, phosphorylation of the histone H2AX occurs and usually be called as ?-H2AX. It is considered as one of the early signals for DSB response. ATM, ATM- and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) are the kinases responsible for the phosphorylation of H2AX. ?-H2AX has a crucial role in amplifying the damaged signal and together with help from other repair factors, the focus is formed and the repair factors access. Moreover, it has been found that methylated histone H3 Lys79 (H3K79) and methylated histone H4 Lys 20 (H4K20) are needed for a focus formation at the DSB damage site, suggesting the importance of histone modification in DSB repair response. How epigenetic marks behave, in terms of the kinetics, following after DSB damage still remain a question in the DSB damage study and it would be the goal of this research.The objective of this study is to understand the histone modifications of DSB repair responses which is important for the alteration of the chromatin structure after DSB damage since the damaged DSB must be in the “open” state for the repair factor to access and repair the damage properly. The relationship between epigenetic marks and other repair factors in DSB repair response is still elusive. This study aims to provide a better understanding of DSB repair and how the epigenetic marks affect the chromatin structure for the repair response.

            In this study, the histone modifications, which are phosphorylation, methylation, acetylation, and ubiquitination will be studied, while ?-H2AX will be used as a DSB repair marker. ATM kinase has catalyzed the reaction of ?-H2AX foci formation and it is essential in DSBs response as a key regulator. ?-H2AX is considered as the crucial event for DSB damage response because it is found to be the trigger of the cascade response and the amplifier of the damaged DNA signal. Therefore, how a cell response to DSB damage in terms of ATM kinase will be investigated in this study and the goal of this research is to understand the kinetics of epigenetics marks that occur after DSB damage by using ?-H2AX foci formation as a marker for DSB damage repair response.How a cell repair from the damaged DSB is an important issue because DSB is a source of a great risk to the cell from a problem of genomic instability which can lead to many diseases such as cancer, Seckel syndrome, or xeroderma pigmentosa. Thus, to be able to understand the mechanism and the kinetics of DSB repair would benefit greatly. The better understanding of how a cell copes with this problem, the more benefit that can be gained which could lead to novel therapeutic approach for the diseases that associated with DSB damage or genomic instability. The kinetics of the repair factors that are involving in DSB repair response has already been reviewed elsewhere but the kinetics of epigenetic marks in DSB repair response has hardly been studied. Therefore, this study aims to investigate the kinetics of the epigenetic marks, which are phosphorylation, methylation, acetylation, and ubiquitination. This study can be combined with other’s studies which were investigated the kinetics of DSB repair factor. Together, the complete model of the mechanism of DSB damage response can be achieved.The goal of this study at the master’s thesis period is to generate a timeline for DSB responses in terms of histone modification changes, which are phosphorylation, methylation, acetylation, and ubiquitination.

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The optimization experiments, which are the optimization of the 405-nm laser diode for DSB induction and screening of hybridoma cells for monoclonal antibody production, will be done by using HeLa cells expressing PCNA-mCherry as a model. ?-H2AX immunostaining will be performed for the selection of hybridoma cells and the observation of these cells will be performed by using a confocal microscope. The fluorescence intensity analysis in hybridoma screening experiments will be studied by using NIS Elements Software program.

The experiments in this study will use ATM+ and ATM- cells as a model for studying the DSB responses in terms of ATM kinase which catalyze the phosphorylation of the histone H2A variant H2AX (?-H2AX) and ?-H2AX will be used as a marker for DSB repair. DSB will be induced in these cells by using a laser microirradiation from the 405-nm laser diode in a confocal microscope which has already been optimized prior to this experiment. The kinetics of histone modification will be tracked by using fluorescence labeled-Fab and the observation will be done by using the same confocal microscope. The fluorescence-labeled Fab will be introduced into ATM+ and ATM- cells by using a bead-loading method which a macromolecule, such as Fab, can be loaded into the cells by the mechanical disruption of the plasma membrane from the glass beads. Further analysis of the DSB induction and repair response, which is cellular alignment and fluorescence intensity measurement that follows after DSB induction, will be studied by using the CellProfiler® cell image analysis software. To confirm the kinetic result of the histone modification changes, RNAi-based technology will be used to knock down the gene of interest and the observation of the DSB repair response will be done by using the same protocol that described above.In the first year, the optimization
of the 405-nm laser diode for DSB introduction
will be performed for the study of DSB damage by observing in
HeLa cells expressing PCNA-mCherry. Then,
the hybridoma cells will be screened by
performing ?-H2AX immunostaining for the clones that can produce monoclonal antibodies that can bind to the
desired antigen and has a low background, thus suited
for the further study.

It has been shown that the histone
methylation, such as H3K79 and H4K20, is crucial for DSB repair by helping to
recruit other repair factors to the DSB damage sites. Therefore, in the first year, the kinetics of histone
methylation and phosphorylation and its relationship with the formation of ?-H2AX
foci will be studied by using ATM+
and ATM- cells as a model because ATM is
the important kinase for the formation of ?-H2AX
foci. The inhibition of other kinases
that catalyze the reaction for ?-H2AX
foci formation, which is ATR and DNA-PK,
will be performed next to confirm the result by using RNAi based technology to
knock down the ATR and PRKDC gene for ATR and DNA-PK,
respectively.It has been shown that
after the inactivation of the human histone acetyltransferases (HATs),
the ATM activation is suppressed, thus suggesting the importance and the
relationship of the acetylation and ATM. Moreover,
after the DSB damage, ubiquitination has been proved to occur rapidly as well. For
example, the polyubiquitination of ?-H2AX
by UBC13/RNF8 ubiquitin ligase complex.
Therefore, in the second year, the roles of acetylation and ubiquitination will
be studied by knocking down the enzymes that catalyze the reaction by using
RNAi based technologies. Together
with the previous results from the first-year
studies, the kinetics of histone modifications after DSB induction can be
generated into a timeline and when
combining the result of this study and another
study on the kinetics of repairing factors in DSB, a better
understanding of the cellular response to DSB damage can be achieved.This study will provide
the timeline for the epigenetic marks change in DSB repair response in terms of
the ATM kinase for the alteration of the chromatin structure which must be in
an “open” state for the repair factors to access and repair the damaged DNA properly.
The knowledge from this study can be used as a preliminary study for the
further analysis of the crosstalk between epigenetics in the DSB repair
response to seeing the relationship
between the epigenetic marks whether to be antagonist or protagonist. Histone
modifications, a key mechanism for the transcriptional regulation, is
reversible and the crosstalk between histone modifications could affect a gene
activation, thus epigenetic marks are the interesting targets for a further
modification to control a gene activation.
The more understanding of the crosstalk between histone modifications could
help for the better manipulation of a gene activation which can be adapted for
the therapeutic purpose of the diseases that are caused by DSB damage. 

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