Jessica Tyler - US grants

? DESCRIPTION (provided by applicant): Packaging the eukaryotic genome into chromatin allows all genomic processes, and consequently growth, development and differentiation, to be highly regulated. This is because our cells use a plethora of mechanisms to change the chromatin structure into a more compact or less compact state, in order to regulate localized access to the genome by the machinery that mediates gene expression, DNA repair, and replication. In the Tyler lab, we study the most profound way that the chromatin structure is changed in the cell, which is removal of the histone proteins from the DNA, termed chromatin disassembly and the opposite process of chromatin assembly. These processes are mediated by histone chaperones together with ATP-dependent chromatin remodelers. Over the years, my group and others have shown that chromatin disassembly and reassembly occurs during replication, gene expression and DNA double-strand break repair. By inactivating the machinery involved in chromatin disassembly and reassembly, we have shown that these chromatin dynamics play an important role in regulating these fundamental genomic processes. However, we still do not know how chromatin disassembly is triggered and how chromatin reassembly occurs in a coordinated fashion at the right time and right place. We will answer these questions by taking advantage of inducible DNA double- strand break systems. It is also important to repress the transcription of genes around a DNA double-strand break in order to achieve DNA repair and to restart transcription after DNA repair is complete. The mechanisms for this are unknown, but we will test the hypothesis that chromatin disassembly and reassembly play important roles in the regulation of transcription inhibition and restart around sites of DNA double-strand damage. Given that the key histone chaperones that mediate these processes, Asf1 and CAF-1, are overexpressed in many types of cancer, this work will not only fill large knowledge gaps but will also have relevance for understanding carcinogenesis.

SUMMARY Cells divide a fixed number of times, termed replicative lifespan, before they stop dividing and senesce. Acceleration of the alterations to biological macromolecules that characterize the normal replicative aging process predisposes us to shortened replicative lifespan and conditions such as progeria. Despite the fundamental importance of the replicative aging process, there are still huge gaps in our understanding of the biological changes that cause aging and the molecular basis of these changes. Given that the mechanisms of aging are highly conserved across eukaryotes, we use the unparalleled genetic power of budding yeast to gain insight into the molecular mechanisms of replicative aging in all eukaryotes. Using the Mother Enrichment Program (MEP) to isolate unprecedented quantities of old cells, we performed a systematic characterization of the replicative aging process in yeast, with the intention of transferring our discoveries to mammalian systems. Using the MEP, my laboratory has been performing a systems biology analysis of the aging process, really for the first time in any organism. Our genome-wide mapping of nucleosome positions uncovered a global loss of nucleosomes during aging, leading to transcriptional upregulation of every gene in the genome. By deep sequencing of the genome during aging we discovered a global increase in retrotransposition, chromosomal translocation, DNA amplification, rDNA instability, transfer of mitochondrial DNA into the nuclear genome and DNA breaks during aging 1. We have now performed metabolomics analysis and ribosome profiling (Ribo-seq) during aging, leading us to our current hypothesis and goal of discovering the molecular details of how protein synthesis changes with aging, the beneficial consequences, and to leverage this information to extend lifespan and healthspan.

SUMMARY Radiation-induced double-strand breaks (DSBs) are fundamental threats to genomic integrity that result in genomic instability if not properly repaired, which can in turn lead to cancer and cell death. Although we know a great deal about the pathways of DSB repair, we know very little about how DSB repair occurs in its natural context in the cell, that is, chromatin. Chromatin by its very nature is an impediment for proteins accessing the DNA, yet the repair machinery is somehow able to navigate through the chromatin and successfully repair DNA damage. Chromatin also plays a key role in transducing the cell's response to DNA damage via the DNA damage cell cycle checkpoint. Until recently, there has been a large gap in our understanding as to how the DSB repair and the DNA damage checkpoint are influenced by the chromatin environment in mammalian cells. Integral to this process is the progression of the DNA repair machinery along a genome that is packaged into chromatin; how this occurs and the influence on the epigenome, has been a long-standing mystery. We have recently shown that chromatin is completely disassembled and reassembled during non-homologous end joining of double-strand DNA breaks in human cells. Excitingly, our recent preliminary data strongly supports an active role for dynamic chromatin assembly onto single stranded DNA (ssDNA) in the midst of homologous recombinational repair of DSBs as an intrinsic step required for DSB repair. Histones occupying ssDNA has never been reported previously in vivo, let alone their playing an important biological role. The proposed studies will uncover the nature of the histone-DNA complexes on ssDNA, and will reveal the elusive mechanism whereby chromatin assembly promotes DSB repair in human cells. By elucidating the mechanism whereby ssDNA-histone complexes contribute to DSB repair, we hope to fill significant gaps in our current knowledge of the chromosomal repair process.

Summary Abstract The overall vision for our research is to discover novel mechanisms by which histone and non-histone proteins on DNA, i.e. chromatin, regulate genomic processes and aging. In particular, we strive to integrate different fields, such as the role of chromatin in genome stability and the role of chromatin in aging. Using a combination of biochemistry, structural biology, molecular genetics in budding yeast, tissue culture and genome-wide approaches, we have discovered that chromatin is disassembled and reassembled during not only gene expression and DNA replication but also during DNA double-strand break repair. We have revealed the mechanistic bases for these events and their key impact on these genomic processes. In more recent years, we have expanded the questions that we address beyond chromatin ? for example uncovering novel mechanistic bases of aging and discovering new ways to extend lifespan. Similarly, inspired by our recent finding that chromatin structure reduces the processing of DNA double-strand breaks to single-strand DNA (termed DNA end resection), we have devised innovative CRISPR/Cas9 gRNA library screening approaches to identify novel activities that regulate DNA end resection during DNA double-strand repair. Most of the cells in the human body are in G0/G1-phase and it is critical that excessive DNA end resection does not occur in these cells. If it were to occur, it would block DNA repair by the only pathway that is used to repair DNA double-strand breaks in G0/G1-phase cells, namely non-homologous end joining (NHEJ), and it would result in translocations and deletions from the ensuing homology-mediated repair. Indeed, the extent of DNA end resection is the critical decision point in the choice between using the NHEJ or homologous recombination (HR) pathway for repairing DNA double-strand breaks. We propose that mechanisms must be in place that limit excessive DNA end resection in G0/G1-phase cells to prevent HR, yet enable sufficient DNA end processing of un-ligatable DNA ends to allow NHEJ-mediated repair. The proteins and pathways that regulate the extent of DNA end resection in G0/G1-phase cells are currently unknown. Thus, a major goal of this program is to discover the machinery and mechanisms that regulate DNA end resection in G0/G1-phase cells. We are uniquely positioned to do this, based on our expertise, novel genetic screening approach and compelling preliminary data. Another critical, yet poorly understood, aspect of genome maintenance is how gene expression is ?shut-off? in the vicinity of a DNA lesion to prevent collisions between the transcription and DNA repair machinery. Similarly, it is crucial that transcription is restarting after DNA double-strand break repair, but the mechanism is unknown. We have recently discovered some of the proteins involved using our novel assays and genetic screens, so the second major goal of this program is to discover the fundamental mechanisms of transcriptional shut-off and restart around DNA double-strand breaks.