Our Research Projects
~ Undergraduate researchers work on projects from three current areas of focus ~
Dissecting the impact of chromatin environment on the DNA damage response in yeast.
DNA double-strand breaks (DSBs) are both beneficial and detrimental to organisms. DSBs are a major driving force for genome diversity and evolution, but they lead to genome instability and accumulation of mutations. The outcome of the DSB response (DDR), which occurs in the context of chromatin, is highly variable depending on its context. The interplay between chromatin structure, histone modification and the DDR is heavily studied, but incompletely understood. Histone modifications and chromatin remodelers have been analyzed in isolation by knocking out the responsible enzymes and observing the effects on the DDR. This approach not only poses potential for indirect effects but also does not address the role of pre-existing chromatin context or combinatorial effects. Differential regulation of DDR in diverse chromatin environments could reveal variability in the stability of chromosomal regions, contributing to patterns of genome rearrangements and evolutionary divergence of genomes. Carefully dissecting the role of pre-existing chromatin will also help form a better foundation for our understanding of the debated de novo chromatin changes that may occur as a consequence of damage signaling- importantly, whether induced changes are context-dependent.
Our hypothesis is that DSBs in different chromatin environments will lead to different outcomes based on the combinatorial effect of their pre-existing and induced histone modifications. This hypothesis is based on findings that link specific histone marks to variable DDR outcomes by disrupting histone modification enzymes. In addition, multiple studies examining chromatin factor recruitment to a single DSB site have provided evidence that damage-induced chromatin changes can drive signaling and guide repair pathways. By systematically producing DSBs in defined genomic regions that represent various combinations of histone modifications and measuring DSB repair, checkpoint activation and apoptosis, while controlling for cutting frequency and cell cycle stage, we can gain insight on specific effects of chromatin on DSB outcome.
DNA double-strand breaks (DSBs) are both beneficial and detrimental to organisms. DSBs are a major driving force for genome diversity and evolution, but they lead to genome instability and accumulation of mutations. The outcome of the DSB response (DDR), which occurs in the context of chromatin, is highly variable depending on its context. The interplay between chromatin structure, histone modification and the DDR is heavily studied, but incompletely understood. Histone modifications and chromatin remodelers have been analyzed in isolation by knocking out the responsible enzymes and observing the effects on the DDR. This approach not only poses potential for indirect effects but also does not address the role of pre-existing chromatin context or combinatorial effects. Differential regulation of DDR in diverse chromatin environments could reveal variability in the stability of chromosomal regions, contributing to patterns of genome rearrangements and evolutionary divergence of genomes. Carefully dissecting the role of pre-existing chromatin will also help form a better foundation for our understanding of the debated de novo chromatin changes that may occur as a consequence of damage signaling- importantly, whether induced changes are context-dependent.
Our hypothesis is that DSBs in different chromatin environments will lead to different outcomes based on the combinatorial effect of their pre-existing and induced histone modifications. This hypothesis is based on findings that link specific histone marks to variable DDR outcomes by disrupting histone modification enzymes. In addition, multiple studies examining chromatin factor recruitment to a single DSB site have provided evidence that damage-induced chromatin changes can drive signaling and guide repair pathways. By systematically producing DSBs in defined genomic regions that represent various combinations of histone modifications and measuring DSB repair, checkpoint activation and apoptosis, while controlling for cutting frequency and cell cycle stage, we can gain insight on specific effects of chromatin on DSB outcome.
Disabling the Interaction Between Rad54 and PCNA to Study its Role in Homologous Recombination
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Rad54 is a multi-functional protein in the process of homologous recombination, a genetic mechanism required for repair of DNA damage and maintenance of genome integrity (i.e., mutation avoidance), immune function, and for producing diversity in sexually reproducing animals. This protein is highly conserved from yeasts to humans, and has many functional domains for its various activities and interactions (Heyer et al., 2006). Although several of these activities of Rad54 are well-characterized, the function of its interaction with the DNA replication clamp PCNA remains poorly understood (Burgess et al., 2013; Zhang et al., 2013). In collaboration with Lumir Krejci at Masaryk University in the Czech Republic, we have been investigating the function of this Rad54-PCNA interaction in the model organism, the yeast Saccharomyces cerevisiae. By mutating the RAD54 gene to render the protein defective for this interaction but leaving all other functional domains intact (rad54-KR/AA), we are testing this protein in multiple assays to determine function of the Rad54-PCNA interaction. This work is significant for the field of homologous recombination because this new interaction links early recombination processes with DNA synthesis. Our results will advance the understanding of how the recombinational repair process is orchestrated in yeast –and given its conservation– in the genomes of all eukaryotes.
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Examining gene expression changes in cis to a double-strand break using fluorescent mRNA reporters
Evidence from mammalian cells in other labs suggest that chromatin compaction at DNA damage sites includes establishment of silent histone methylation marks propagated for tens of kilobases from a double-strand break site and repression of gene expression proximal to DNA breaks. Transcriptional repression in cis to a DSB has been observed in yeast as well, but could be due to possible confounding DSB end processing events and deserves careful study. Overall, the results from higher eukaryotes suggests that there are local break-induced changes to the chromatin fiber that transmit information about nearby damage to the transcriptional machinery, but this remains to be shown in yeast. We have created a PFK1-MS2 fusion strain to investigate transcription changes in the presence of double strand breaks and to serve as a real-time reporter.
Evidence from mammalian cells in other labs suggest that chromatin compaction at DNA damage sites includes establishment of silent histone methylation marks propagated for tens of kilobases from a double-strand break site and repression of gene expression proximal to DNA breaks. Transcriptional repression in cis to a DSB has been observed in yeast as well, but could be due to possible confounding DSB end processing events and deserves careful study. Overall, the results from higher eukaryotes suggests that there are local break-induced changes to the chromatin fiber that transmit information about nearby damage to the transcriptional machinery, but this remains to be shown in yeast. We have created a PFK1-MS2 fusion strain to investigate transcription changes in the presence of double strand breaks and to serve as a real-time reporter.