Our long-term goal is to define connections between replication stress and epigenetic states and processes in human cells. The importance of this research domain to human health is underscored by the notion that epigenetic changes are expected to be more easily reversible than a genetic mutation or deletion, can thus they can hold greater potential for therapeutic manipulation. Replication stress is a state of genomic replication characterized by abnormal density, distribution, and stability of replication forks. Replication stress can be triggered by chemotherapy. As a source of genomic instability it is also implicated in early steps of carcinogenesis. The important questions that emerge from recent studies are whether epigenetic factors modulate cellular resistance to replication stress, and conversely, whether replication stress can challenge or compromise epigenetic inheritance thus opening another avenue to cellular degeneration or transformation. We will address these questions by dissecting a specific problem: chromatin modification and remodeling around moving and stalling replication forks and its roles in the context of a deficiency in the RECQ helicase WRN, mutated in the Werner syndrome of premature aging. On the one hand, recent work now implicates WRN in chromatin maintenance and epigenetic stability. On the other hand, we have previously shown that WRN absence compromises cellular resistance to replication stress and, more recently, that histone deacetylases HDAC1 and 2 cooperate with WRN in counteracting replication fork inactivation during the replication stress caused by nucleotide pool depletion. With the aim of a greater understanding of the connections between altered replication, altered chromatin, and the cellular biology of WRN deficiency, we will determine the mechanism of cooperation between WRN and HDACs in the context of nascent chromatin maturation and epigenetic changes occurring during and after replication stress in normal and WRN-deficient cells, and their effect on cell survival, proliferation, and lifespan. Our approach integrates standard cellular biology assays with high-resolution, functional analyses of genomic replication in vivo at DNA and protein levels (respectively, microfluidic-assisted replication track analysis or ma-RTA, and immunoprecipitation of nascent DNA, or iPOND). We will use RNAi and CRISPR/Cas9 manipulation to inactivate expression of the genes of interest. We will also use both targeted and unbiased approaches to identify the factors and processes involved in the functional interaction between WRN and HDAC1 and 2. We will query for specific protein candidates' involvement as well as perform mass spectrometric analyses of proteins associated with stalled replication forks in WRN or HDAC1,2-deficient and normal cells. We will also use a previously validated pipeline to perform a siRNA screen for epigenetic modifiers of HDAC, WRN- dependent replication stress phenotypes.

Public Health Relevance

The work of the human genome is regulated on many levels, and recent studies increasingly point to the role of epigenetics - the 'bookmarks' left on the nucleotide sequence 'text' of the genome - as critical in determining how cells grow, divide, adopt their fate in the human body, and react to environment as part of an organism. We want to contribute to the developing area of research concerned with understanding of how epigenetics helps cells faithfully replicate their genetic information on during cell division on te one hand, and how faulty replication may signal epigenetic change, on the other. We will follow up on our finding that connects one of the guardians of genomic stability, the Werner syndrome protein, with the proteins that control how epigenetic bookmarks are reproduced when a cell copies its genome prior to dividing into two cells

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Reddy, Michael K
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University of Washington
Schools of Medicine
United States
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Oshima, Junko; Sidorova, Julia M; Monnat Jr, Raymond J (2017) Werner syndrome: Clinical features, pathogenesis and potential therapeutic interventions. Ageing Res Rev 33:105-114
Sidorova, Julia (2017) A game of substrates: replication fork remodeling and its roles in genome stability and chemo-resistance. Cell Stress 1:115-133
Kehrli, Keffy; Phelps, Michael; Lazarchuk, Pavlo et al. (2016) Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest. J Biol Chem 291:24487-24503
Welcsh, Piri; Kehrli, Keffy; Lazarchuk, Pavlo et al. (2016) Application of the microfluidic-assisted replication track analysis to measure DNA repair in human and mouse cells. Methods 108:99-110