Damage in the DNA inhibits transcription of genes by RNA polymerase II, which copies the genetic information of DNA into RNA. This impediment of RNA polymerase II results in severe cellular dysfunction and accelerated aging. Through a consortium combining unique complementary knowledge and expertise, we can study for the first time the causes and consequences of DNA damage from the perspective of a single molecule to that of a whole organism. Using this approach, we will study what exactly happens to RNA polymerase when encountering DNA damage, and directly link this to the consequences at the cellular and organism level.

The mammalian genome is a highly organized structure where distinct chromosomal domains occupy discrete territories and position in a non-random fashion. Genome organization also determines the proper acquisition of various biological processes, including DNA replication. Errors in DNA replication can catastrophically degrade both the genetic and the epigenetic integrity of chromosomes, contributing to genome instability that drives tumorigenesis. Chromatin modifiers are the major determinants of chromatin architecture as well as accessibility to nucleosomal DNA which allows efficient DNA replication. However, the mechanistic understanding of 3-D nuclear chromatin organization orchestrated via chromatin modifiers in mediating stabilization of replication machinery upon replication stress has been poorly studied. My hypothesis is that stalled replication forks, especially at the regions prone to acquire breaks, undergo compartmentalization/re-organization within 3-D nuclear space in response to replication stress to allow efficient fork protection and fork restart ability. My lab uses a multidisciplinary approach for molecular characterization of DNA replication stress in mammalian system, taking advantage of a unique combination of specialized single molecule analysis of replication forks, super-resolution/high-throughput imaging and unique 3-D chromatin conformation capture techniques. Building on my expertise in the study of chromatin organization and DNA replication stress, for this proposal, I will focus on dissecting the role of chromatin architecture in tolerance to replication stress by: a) developing a high-resolution technology to capture novel chromatin interactions ensuing directly at replicating sites in response to replication stress; b) determine the role of chromatin modifiers in mobilizing the genomic loci under replication stress; and c) unravel the significance of re-organization of chromatin architecture in developing resistance towards replication stress in cancer cells. These studies will contribute to an overall understanding of how chromatin architecture adaptations in diseases such as, cancer can develop resistance towards replication stress inducing chemotherapeutic drugs.

Hereditary defects in the essential and multifunctional transcription and DNA repair factor TFIIH are associated with several distinct, clinically heterogeneous diseases characterized by cancer predisposition, (progressive) neurological defects, developmental failure and segmental progeria, whose pathogenesis is not fully understood. The function and activity of TFIIH has been thoroughly investigated, but it is still not entirely clear how mutations in the same complex can lead to diverse symptoms and human diseases and why the impact of hereditary mutations differs depending on the tissue type. Systematic comparison of TFIIH mutations in cells is difficult because TFIIH is essential and patient cells are not isogenic and often compound-heterozygote. To better understand mutant TFIIH activity in cells, in this project we propose to generate and functionally compare multiple patient-derived mutations in the same TFIIH gene, in isogenic human cells and in C. elegans as in vivo model. To this end, we will label, by knock-in, endogenous TFIIH with the multi-purpose GFP-tag while simultaneously introducing patient-derived mutations in its XPD helicase subunit, using CRISPR-Cas9. Next, using state-of-the-art DNA repair assays, live cell confocal imaging, proteomics and genomics approaches, we will thoroughly investigate the activity of each mutant TFIIH in nucleotide excision repair and transcription, in relation to its phenotypic impact, in both human cells in culture and in vivo, in C. elegans, to study mutant TFIIH mechanism and impact in differentiated cell types such as neurons. As functional TFIIH is essential for life and because its dysfunction causes developmental failure, cancer and aging, it is of major importance to understand its precise activity to be able to promote human health.

Breaking both strands of DNA is a serious threat to the encoded information. Break repair requires a whole team of proteins self-assembled into machinery in the complex environment of the cell nucleus and in competition with other genome processing events. To understand how this works we developed new tools to visualize the organization of DNA repair proteins in living cells. These tools allow to determine the contributions of different repair factors to the assembly of proteins at DNA damage in cells required for faithful DNA repair. These tools contribute to new directions for cancer therapy and optimizing CRISPR/Cas genome editing.

Chromatin modifiers are the major determinants of accessibility to nucleosomal DNA which allows for the accomplishment of essential biological processes like DNA replication and repair in the most efficient manner. Errors in DNA replication can catastrophically degrade both the genetic and the epigenetic integrity of chromosomes, contributing to a state of genome instability that drives tumorigenesis. Many commonly used anti-cancer drugs interfere with the DNA replication process to enhance the intrinsic genomic instability of cancer cells to cause fatal DNA damage, but modulation of the replication machinery can also result in chemoresistance to these drugs. Mounting evidences have revealed the high mutation/amplification rate in chromatin modifier genes in several types of cancers that possibly contribute to build tolerance towards replication stress inducing drugs. However, mechanisms by which chromatin modifying activity responds to enhanced stress to stabilize the replication machinery and the cellular factors involved in the process are poorly understood. Using a quantitative proteomic approach, I have identified a highly conserved histone modifying factor, EHMT2/G9a to be associated with replication forks. The decreased expression of EHMT2/G9a results in increased sensitivity towards DNA replication stress-inducing chemotherapeutic drugs in BRCA1-deficient tumor cells. These preliminary data strongly suggest that changes to local epigenetic marks on histones in the vicinity of replication fork regulates replication fork dynamics and prevents genome instability. To test this hypothesis, I will use a combination of quantitative proteomics, super-resolution microscopy, specialized single molecule techniques and genomics. My expertise in using these combinations of technologies will reveal the intricate molecular mechanisms underlying the role of chromatin modifications in the context of DNA replication stress and its role in genome stability.