Genome Integrity Discussion Group June 2013
Monday, June 3, 2013
The New York Academy of Sciences
Presented by the Genome Integrity Discussion Group
The greater New York Metropolitan area has become a leading center for research on chromosome biology and function, as well as for research at the interface between chromosome integrity and onset and progression of malignancy. The connection between cancer and genome integrity is widely appreciated, and the concentration of excellence in this field is unparalleled anywhere in the world. The Genome Integrity meetings are designed to provide a forum for interactions between the many basic science and clinically-oriented research groups working on these issues. We feel that these interactions will not only facilitate synergy between labs, but also provide a context in which previously unappreciated complementarities will be revealed.
In that spirit, the talks will cover a broad range of areas, including, but not limited to the DNA damage response and cancer predisposition, DNA replication, transcription, chromatin modification, recombination, cell cycle control, telomeres, chromosome segregation, epigenetic states, as well as the emergence of new technologies relevant to research in genome integrity. Although a primary focus is upon basic mechanisms and processes, these areas are pertinent to cancer and myriad human disease states, and it is expected that this will be reflected in the substance of our discussions.
Genome Integrity Discussion Group meetings are organized under the leadership of John Petrini (Memorial Sloan Kettering Cancer Center), and Rodney Rothstein and Lorraine Symington (Columbia University). The year-end meeting includes a scientific symposium with a keynote presentation from 1:00 to 4:00 PM, followed by a poster session and networking reception from 4:00 to 5:30 PM.
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* Presentation titles and times are subject to change.
Monday June 3, 2013
Keynote address: Defective DNA Strand Break Repair and Links to Human Disease
Coffee break and poster set-up
The role of the LINC complex in the 53BP1-driven mobility and NHEJ of dysfunctional telomeres
Mph1 interplay with nucleases Mus81-Mms4, Yen1 and Rad1-Rad10 to prevent reciprocal translocations between dispersed repeats
Epe1 recruits BET family bromodomain protein Bof1 to establish heterochromatin boundaries
Cell cycle transitions in the absence of nucleosomes
Poster Session and Networking Reception
John Petrini, PhD
Memorial Sloan-Kettering Cancer Center
Rodney Rothstein, PhD
Columbia University Medical Center
Lorraine Symington, PhD
Columbia University Medical Center
Stephen West, PhD
London Research Institute, Cancer Research UK
Stephen West received his PhD in biochemistry from Newcastle University, England, before joining the Department of Therapeutic Radiology at Yale in 1978. There, he was a post-doc with Paul Howard-Flanders, one of the early pioneers in the field of DNA repair. Steve moved back to the UK in 1985 to set up a laboratory at the Imperial Cancer Research Fund (now Cancer Research UK) where he is Senior Group Leader and Deputy Director of Clare Hall Laboratories. Steve's work has focused on the mechanisms of DNA repair by homologous recombination, and the links between repair defects, genome instability and cancer. His early work led to an understanding of homologous pairing by RecA and RAD51, and the resolution of recombination intermediates. Steve discovered the first cellular Holliday junction resolvase (E. coli RuvC), the bacterial branch migration complex (RuvAB), and the human GEN1 Holliday junction resolvase. His work currently revolves around the molecular functions of the BRCA2 tumour suppressor, and the roles and interplay of various Holliday junction resolvases that process recombination intermediates in human cells. Steve has received many awards for his scientific achievements. These include election to EMBO (1994), as a Fellow of the Royal Society (1995), the Academy of Medical Sciences (2000), and the European Academy of Cancer Sciences (2011). He has also received several prizes, including the Swiss Bridge Prize for Cancer Research (2001 and 2009), the Leeuwenhoek Medal of the Royal Society (2002), the Louis-Jeantet Prize for Medicine (2007), the Novartis Medal from the Biochemical Society (2008), the GlaxoSmithKline Medal of the Royal Society (2010), and most recently the Genetics Medal (2012).
de Lange lab, Rockefeller University
Symington lab, Columbia University Medical Center
Jia lab, Columbia University
Christian Zierhut, PhD
Funabiki lab, Rockefeller University
Defective DNA Strand Break Repair and Links to Human Disease
Stephen West, PhD, London Research Institute, Cancer Research UK
All living organisms feature DNA repair pathways that safeguard the integrity of the genome, and mutations in proteins that mediate key events in DNA repair have been linked to genome instability and tumourigenesis. Homologous recombination provides an important DNA repair pathway that is needed for the restoration and restart of broken replication forks, for the rejoining of chromosome breaks, and for the processing of DNA crosslinks. Mutations in genes that encode a variety of recombination proteins are linked to breast cancers and to inheritable diseases such as Blooms Syndrome (BS) and Fanconi Anemia (FA). In recent work, we purified the BLM protein (defective in BS), the BRCA2 (FANCD1) and PALB2 (FANCN) tumour suppressors (mutated in some cases of FA), and the newly discovered FANCP protein, also known as SLX4, and have initiated structure-function analyses to elucidate their molecular functions. How these proteins process DNA, and how they are regulated and controlled to direct the outcome of recombinational repair, is now revealing unexpected insights that extend our understanding of efficient DNA repair and tumour avoidance.
The Role of the LINC Complex in the 53BP1-Driven Mobility and NHEJ of Dysfunctional Telomeres
Francisca Lottersberger, PhD, de Lange lab, Rockefeller University
The removal of the shelterin component TRF2 from the telomeres elicits an ATM-dependent DNA damage response that leads to NHEJ-mediated telomere fusions. A key role in this repair pathway is played by the DNA damage factor 53BP1, which is recruited at deprotected telomeres in an ATM-dependent manner, promotes NHEJ, increase telomere mobility and inhibits 5’ end resection (Dimitrova et al., 2008; Lottersberger et al, 2013; Zimmerman et al., 2013). Here we address the mechanism by which 53BP1 enhances the mobility of dysfunctional telomeres. Treatment with nocodazole and low concentrations on vincristine, which depolymerize microtubules, inhibited the 53BP1-driven mobility of dysfunctional telomeres, whereas the microtubule stabilizing drug taxol had no effect. The effect of nocodazole was readily reversible upon wash-out of the drug. This indicated that telomere mobility dependent on a microtubule mediated movement. We therefore examined the role of the LINC (LInker of the Nucleoskeleton and Cytoskeleton) complex, which was previously implicated in telomere movement movement in meiosis in S. pombe and mammals (Chikashige et al., 2006; Ding et al., 2007; Schober et al., 2009). Using SV40 immortalized TRF2 conditional knockout MEFs we tested shRNAs to various LINC components for their effect on telomere fusions, making the assumption that the mobility of dysfunctional telomeres is required for efficient fusion. These experiments implicated the inner nuclear envelope proteins SUN1 and SUN2, the KASH domain protein Nesprin-4, as well as kinesin-1 and kinesin-2 in the 53BP1-driven mobility of dysfunctional telomeres. Recent live-cell imaging of dysfunctional, TRF2-depleted telomeres in conditional TRF2/SUN1/2 double knockout cells indicates that SUN1 and SUN2 are indeed required for the movement of dysfunctional telomeres. Thus, 53BP1 mediates the movement of dysfunctional telomeres by promoting their interaction with the LINC complex and allowing their kinesin-mediated movement along microtubules.
Mph1 Interplay with Nucleases Mus81-Mms4, Yen1 and Rad1-Rad10 to Prevent Reciprocal Translocations between Dispersed Repeats
Gerard Mazón, PhD, Symington lab, Columbia University Medical Center Homology-dependent repair of double-strand breaks (DSBs) from non-sister templates has the potential to generate deleterious genome rearrangements. We show how the Mph1 helicase prevents crossovers between ectopic sequences by removing substrates for Mus81-Mms4 or Rad1-Rad10 cleavage while a role for Yen1 is only detected in the absence of Mus81. Cells lacking Mph1 and the three nucleases are highly defective in the repair a single DSB, suggesting the recombination intermediates that accumulate cannot be processed by the Sgs1-Top3-Rmi1 complex (STR). Consistent with this hypothesis, ectopic joint molecules accumulate transiently in the mph1Δ mutant and persistently when Mus81 is eliminated. Furthermore, the ectopic JMs formed in the absence of Mus81 are connected by a single HJ explaining why STR is unable to process them. We suggest that Mph1 and Mus81-Mms4 recognize a common early strand exchange intermediate and direct repair to non-crossover or crossover outcomes, respectively.
Epe1 Recruits BET Family Bromodomain Protein Bof1 to Establish Heterochromatin Boundaries
Jiyong Wang, PhD, Jia lab, Columbia University
Heterochromatin spreading leads to the silencing of genes within its path, and boundary elements have evolved to constrain such spreading. In fission yeast, heterochromatin at centromeres I and III is flanked by inverted repeats termed IRCs, which are required for proper boundary functions. However, the mechanisms by which IRCs prevent heterochromatin spreading are unknown. Here, we identified Bof1, which is homologous to the mammalian BET family double bromodomain proteins as a factor required for proper boundary function at IRCs. Bof1 is enriched at IRCs through its interaction with the boundary protein Epe1. The bromodomains of Bof1 recognize acetylated histone H4 tails and antagonize Sir2-mediated deacetylation of histone H4K16 to prevent heterochromatin spreading. The conserved BET (bromodomain extra terminal) family of bromodomain proteins in humans is the focus of drug-mediated epigenetic cancer treatment. Although they are implicated in regulating oncogene Myc expression, the precise mechanisms of these inhibitors are unknown. Our analyses reveal a novel mechanism of heterochromatin boundary formation through the direct recruitment of a histone tail binding protein to specific DNA element, raising the possibility that BET inhibitors potentially affect the spreading of heterochromatin and reset the epigenetic landscape in cancer cells.
Cell Cycle Transitions in the Absence of Nucleosomes
Christian Zierhut, PhD, Funabiki lab, Rockefeller University
Eukaryotic nuclear DNA is packaged into large structures known as chromatin. This structure’s fundamental unit is the nucleosome, formed by DNA wrapped around the histone proteins H2A, H2B, H3 and H4. By limiting accessibility to DNA, nucleosomes may act inhibitory to many processes, but by directly recruiting proteins they may also act stimulatory. Nucleosomes are thus intimately connected to all aspects of chromosome biology. However, because of the essential nature of histones and their extremely long half-lives, these effects are hard to analyse in genetic model organisms. Furthermore, histones are important regulators of transcription, and therefore pleiotropic transcriptional changes cannot be excluded following histone manipulations. Consequently, most evidence for the functions of histones in DNA biology is correlative. Using a novel approach to study transcription-independent functions of nucleosomes, we have analysed the contributions of nucleosomes to mitosis, nuclear envelope formation, DNA damage checkpoint signalling and DNA damage processing/repair. We have also analysed a number of key cell cycle proteins that are associated with chromosomes for whether they are affected by the absence of nucleosomes. To this end, we have developed a method to deplete H3 and H4 fromXenopus laevis egg extracts that faithfully recapitulate chromosome physiology in the absence of transcription, rendering the extracts incapable of nucleosome formation. We found that DNA damage checkpoint signaling in response to double-stranded DNA breaks (DSBs) is not affected by nucleosomes at a low number of breaks but is stimulated if a high number of breaks are present. Preliminary results further suggest that nucleosomes inhibit DSB resection, thus stimulating repair by non-homologous end joining. Mitotic spindle formation was found to depend on nucleosomes, but membrane association after mitosis does not. In contrast, incorporation of nuclear pore proteins following mitosis depends on nucleosomes. This can be attributed to a defect in the generation of a chromatin-bound intermediate in nuclear pore formation on nucleosome-free DNA, as shown by the absence of the nucleoporin ELYS from non-nucleosomal DNA both in extract and in vitro. As ELYS is the first nucleoporin to bind chromosomes following mitosis, being required for the recruitment of most other nucleoporins, these results thus suggest that recognition of nucleosomes by ELYS triggers the formation of nuclear pores upon exit from mitosis. Together, our results comprise the first study of how chromatin as a whole directly affects chromosome physiology, and allow us to generate a framework for nucleosome functions throughout the cell cycle.
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