Genome Integrity Discussion Group December 2014

Agenda

* Presentation titles and times are subject to change.

Abstracts

Recruitment and Spreading of Condensin Complexes in C. Elegans
Sevinc Ercan, PhD, Department of Biology, New York University

Condensins are multi-subunit protein complexes that are essential for chromosome condensation during mitosis and meiosis, and play key roles in transcription regulation during interphase. Metazoans contain two condensins (I and II), which perform different functions and localize to different chromosomal regions. C. elegans contains a third condensin (DC) that is targeted to and represses transcription of the X chromosome for dosage compensation. Our analyses of condensin I, II, and DC binding in C. elegans show that condensins bind to a subset of active promoters, tRNA genes and putative enhancers, as well as unannotated intergenic sites. The mechanisms by which condensins are targeted to their genomic binding sites remain unclear. Our work indicates that condensin binding is established in two-steps: recruitment and spreading. Recruitment is DNA sequence specific and determined in part by sequence motifs that may be bound by recruiter proteins. Spreading is not sequence specific and occurs onto a subset of active promoters and other intergenic sites. Here, we will present the results form our ectopic recruitment studies using condensin DC, to understand the factors involved in X–specific recruitment. We will also briefly discuss condensin DC spreading, which increases the level of H4K20me1, a mitosis associated histone modification, and this results in transcription repression.

Loss of ATRX in ALT suppresses resolution of telomere cohesion to control recombination
Susan Smith, The Skirball Institute, New York University School of Medicine, New York, NY

The chromatin-remodeling factor ATRX is the protein most frequently lost in tumors that use the recombination based, alternative lengthening of telomeres (ALT) mechanism for telomere maintenance, but its role in telomere recombination is not known. We found that loss of ATRX suppresses resolution of sister telomere cohesion at mitosis. In the absence of ATRX, the histone variant macroH2A1.1 binds to the PARP tankyrase 1, preventing it from localizing to telomeres and from resolving cohesion. Restoration of sister telomere resolution by overexpression of tankyrase 1 (or the macroH2A1.1-binding domain of ATRX) results in rampant recombination between non-sister telomeres, genomic instability, and impaired cell growth, indicating that keeping sister telomeres in close proximity into mitosis is essential for the ALT cell state. The newly identified ATRX-macroH2A1.1-tankyrase axis may provide a novel therapeutic target in ALT tumors.
 
Coauthor: Mahesh Ramamoorthy, The Skirball Institute, New York University School of Medicine, New York, NY

Mechanism of DNA Interstrand Crosslink Processing by Repair Nuclease FAN1
Renjing Wang¹

DNA interstrand crosslinks (ICLs) are highly toxic lesions associated with cancer and degenerative diseases. ICLs can be repaired by the Fanconi Anemia (FA) pathway and through FA-independent processes involving the FAN1 nuclease. Here, FAN1-DNA crystal structures and biochemical data reveal that human FAN1 cleaves DNA successively at every third nucleotide. In vitro, this exonuclease mechanism allows FAN1 to excise an ICL from one strand through flanking incisions. DNA access requires a 5’-terminal phosphate anchor at a nick or 1-2 nucleotide flap, and is augmented by a 3’ flap, suggesting FAN1 action is coupled to DNA synthesis or recombination. FAN1’s mechanism of ICL excision is well suited for processing other localized DNA adducts as well.
 
Coauthors: Nicole S. Persky¹, Barney Yoo², Ouathek Ouerfelli², Agata Smogorzewska³, Stephen J. Elledge^4,5, Nikola P. Pavletich¹
 
1. Structural Biology Program and Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
2. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
3. Laboratory of Genome Maintenance, The Rockefeller University, New York, NY 10065, USA
4. Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
5. Division of Genetics, Brigham and Women’s Hospital, Boston, MA 02115, USA

Visualizing Dynamic Kinetochore Structure Using Super-Resolution Microscopy
David J. Wynne, Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, NY 10065, USA

During chromosome segregation, the function of kinetochores changes from interacting with the lateral surfaces of microtubules and activating the spindle assembly checkpoint (SAC) to capturing microtubule ends and silencing the SAC. The molecular mechanism that couples this functional change to microtubule attachment status remains unclear. Although the molecular identity of kinetochore components has now been well established, we have just begun to understand the assembly process and spatial arrangement of this dynamic machine.  Applying 3-D super-resolution imaging to Xenopus egg extracts, we reveal that the kinetochore is spatially and functionally segmented into a stable core module supporting end-on attachment and an expandable module responsible for lateral attachment and SAC signaling. Unexpectedly, the inner kinetochore component CENP-C is an integral component of the expandable module, whose assembly also depends on outer kinetochore proteins (Bub1, BubR1) and multiple protein kinases (Aurora B, Haspin, Plx1, Mps1) and is suppressed by protein phosphatase 1.  We propose that the expandable module consists of a phosphorylation-dependent copolymer that spatially segregates kinetochore functions to help couple end-on attachment and SAC silencing.
 
Coauthor: Hironori Funabiki, Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, NY 10065, USA

RNA-programmed genome reorganization in the ciliate Oxytricha
Laura Landweber, Princeton University, Princeton NJ

The ciliate Oxytricha possesses a dynamic pair of genomes, and massive DNA rearrangements produce a highly fragmented but functional somatic genome from a complex germline genome. This process eliminates nearly all noncoding DNA, including transposons, and rearranges over 225,000 short DNA segments to produce tiny gene-sized "nanochromosomes." In the precursor germline genome, the shattered segments of different genes often interweave with each other, frequently overlap and sometimes combinatorially assemble (Chen et al. 2014 Cell 158:1187). The whole process produces a mature, somatic genome of over 16,000 nanochromosomes (Swart et al., 2013 PLoS Biology 11: e1001473).  Noncoding RNAs regulate the entire process of genome rearrangement. Maternally-inherited, long, non-coding (lnc) RNAs provide three layers of continuity across generations, including serving as templates for both genome remodeling and RNA-guided DNA repair (Nowacki et al., 2008 Nature 451:153) while also regulating gene dosage and chromosome copy number (Nowacki et al., 2010 PNAS 107:22140). This illustrates the ability of lncRNAs to transmit heritable changes to the next generation. Furthermore, 27nt piRNAs provide the critical information to mark and protect the retained DNA segments of the genome (Fang et al., 2012 Cell 151:1243). Together, Oxytricha's elaborate epigenome, assembled through complex interacting networks of both long and small non-coding RNAs, encapsulates an RNA-driven world packaged in a modern cell. The mechanism for all of these dynamic actions bypasses the traditional modern pathway of inheritance via DNA, hinting at the power of RNA molecules to sculpt genomic information.

The regulation of genome replication
Lisa Hang, Wei Tan, Xiaolan Zhao, Department of Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, USA

DNA replication is a highly regulated process that is essential for preserving the integrity of the genome. To cope with endogenous and exogenous sources of replication stress, a network of regulatory factors is evolved to facilitate synthesis over damaged or blocked template DNA.  Some are DNA metabolism enzymes that physically remove lesions or blocks from templates. Others are protein modification enzymes that can change the properties of many proteins at once to elicit changes in favor of replication. A third group is scaffolds that can establish specific molecular associations with enzymes or other factors that are required for overcoming replication obstacles. How each of these factors functions during replication and how they coordinate are important questions to understand in order to decipher the sophisticated and multi-facets replication regulation. We will present some current findings from our lab using the budding yeast as a model system to address these questions.

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