Genome Integrity Discussion Group June 2015

Agenda

* Presentation times are subject to change.

Abstracts

Sae2 and RPA Collaborate to Prevent Palindromic Gene Amplification
Sarah K Deng¹

A large number of diverse and complex genomic rearrangements have been observed, in cancer and other human diseases. A common type of gross chromosomal rearrangement (GCR) is gene amplification, an increase in gene copy number. Duplicated sequences are clustered segmentally within a genomic region with extra copies organized as both direct (head to tail) and inverted (tail to tail) duplications. Interestingly, inverted duplications (also called palindromic duplications) have been identified in metastatic pancreatic cancer and ErbB-2 (HER2) positive breast cancers. Thus, understanding their mechanism of formation advances our understanding of GCR formation with important implications for human health. Foldback priming at DNA double-stranded breaks is one of several mechanism proposed to initiate palindromic gene amplification. Using a Saccharomyces cerevisiae assay to detect gross chromosomal rearrangements (GCRs), we found that GCRs from sae2Δ and nuclease-defective mre11-H125N derivatives were predominantly palindromic duplications. Short inverted repeats were identified at the breakpoints, consistent with intra-strand annealing at a spontaneous double-strand break to create a foldback that is stable in the absence of Sae2 or the Mre11 nuclease. The frequency of inverted duplications was elevated ~1000-fold in the hypomorphic rfa1-t33 sae2Δ mutant. Furthermore, 30% of the inverted duplications recovered from the rfa1-t33 sae2Δ mutant had sequences adjacent to the breakpoint duplicated multiple times, similar to higher order amplifications observed in a subset of cancers. Most of the inverted duplications were associated with a duplication of a second chromosome region bounded by a repeated sequence and a telomere suggesting the hairpin-capped chromosome is replicated to form a dicentric isochromosome that is broken and subsequently repaired by homologous recombination, using interspersed repeats, to form a stable monocentric chromosome. We propose secondary structures within ssDNA are potent instigators of genome instability, and RPA and Sae2 play important roles in preventing their formation and propagation.
 
Coauthors: Yi Yin², Thomas D. Petes² and Lorraine S Symington¹
1 Columbia University Medical Center
2 Duke University Medical Center
 

Effect of BRCA Mutations on Homology-Directed Repair During Mouse Mammary Gland Development
Elizabeth M. Kass¹ Defects in homology-directed repair (HDR)—considered the most accurate of the three major pathways for repairing double-strand breaks (DSBs) in DNA in mammalian cells—can lead to genomic instability and are associated with tumor predisposition. Mutations in multiple genes involved in HDR are linked to breast cancer susceptibility including BRCA1 and BRCA2. The mammary gland is highly influenced by its hormonal environment and undergoes significant changes during puberty, pregnancy and involution, but how these developmental changes impact DSB repair and why mutations in canonical HDR proteins increase susceptibility to mammary tumorigenesis are uncertain.
 
A widely used reporter for HDR is DR-GFP in which direct repeats of two defective GFP genes are induced to recombine by I-SceI endonuclease cleavage of one of the repeats, resulting in a functional GFP gene. We have recently generated DR-GFP mice that express I-SceI under the control of a tetracycline-inducible promoter, allowing for the analysis of HDR within both primary cells and the tissues themselves.
 
To gain insight into the effects of breast cancer predisposing mutations on HDR in primary mammary epithelial cells, the I-SceI/DR-GFP mice were crossed to mice carrying hypomorphic mutations in Brca1 (Brca1tr/tr) or Brca2 (Brca2ex27/ex27). An approximately 2-fold reduction in HDR was observed in mammary epithelial cells from virgin Brca1tr/tr I-SceI DR-GFP mice compared to littermate controls, while a 6-fold reduction was observed in primary mammary epithelial cells from virgin Brca2ex27/ex27 mice. HDR was similarly reduced in ear fibroblasts from these animals, suggesting the DSB repair defects in primary mammary epithelial cells from BRCA mutant virgin mice are not tissue specific.
 
To understand how DSB repair processes in the mammary gland are affected by developmental changes, we are assessing HDR at different stages of mammary gland development in vivo. A significant increase in HDR relative to overall DSB repair was observed in mammary tissue from mice at mid-pregnancy compared to virgin littermates. Interestingly, our preliminary results indicate that both the Brca1tr and Brca2ex27 mutants also show increased HDR during pregnancy, such that the relative HDR reduction compared to wild-type littermates is significantly less pronounced in both mutants during pregnancy, suggesting developmental stage-specific factors may influence HDR proficiency even in repair mutants.
 
Coauthors: Mary Ellen Moynahan² and Maria Jasin¹
Developmental Biology Program¹ and Department of Medicine², Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA

A Chemical Proteomics Approach Reveals a Direct Interaction between 53BP1 and gH2AX Involved in the DNA Damage Response
Ralph E. Kleiner, PhD, Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, New York, United States

Post-translational modifications on histone tails can regulate the localization of chromatin-associated proteins implicated in essential cellular processes. These modifications are often sub-stoichiometric and mediators of low-affinity interactions, which makes identifying their ‘readers’ challenging. In particular, the efficient repair of DNA double-strand breaks involves the phosphorylation of the histone variant H2AX (‘gH2AX’), which accumulates in foci at the site of damage. In current models, the recruitment of multiple DNA repair proteins to gH2AX foci depends on direct recognition of this mark by a single protein, MDC1 (mediator of damage checkpoint 1). However, DNA repair proteins can accumulate at gH2AX foci without MDC1, suggesting that other gH2AX ‘readers’ exist. Here, we use a chemical proteomics approach to profile direct and phospho-selective binders of gH2AX in native proteomes. We identify proteins that ‘read’ gH2AX, including the DNA damage response mediator, 53BP1, which we show interacts with this ‘mark’ through its BRCT domains. Additionally, we replace endogenous 53BP1 with wild type or a mutant form with reduced gH2AX affinity and investigate the functional relevance of this interaction in the context of endogenous and exogenous DNA damage. Our results show how direct recognition of gH2AX can modulate protein localization at DNA damage sites, and suggest how specific chromatin ‘mark’-‘reader’ interactions contribute to essential mechanisms that ensure genome stability.

Fate of Dicentric Chromosomes Formed through Telomere Fusion
John Maciejowski, Rockefeller University, New York, New York, United States

Telomere crisis is thought to fuel genome instability through breakage-fusion-bridge cycles initiated when telomeres fuse and generate dicentric chromosomes. Dicentric chromosomes are widely assumed to break during anaphase or cytokinesis. Because calculations of the spindle force relative to the tensile strength of DNA indicate that anaphase breakage is unlikely, we examined the fate of dicentrics formed through telomere fusions. 
 
Using spinning disk microscopy of large fields imaged at 63X (using ‘stitching’), we followed the fate of large numbers of RPE1-hTERT cells lacking Rb and p21 as they were undergoing telomere fusions in response to conditional inactivation of the shelterin protein TRF2. Using an H2B-mCherry chromatin marker to follow cells with overt telomere fusions, we find that dicentric chromosomes do not break in anaphase or during cytokinesis. Instead, most (if not all) dicentric chromosomes form persistent chromatin bridges that connect daughter nuclei well into the next cell cycle before their ultimate rupture. Daughter cells can move apart by as much as 300 microns while maintaining a chromatin bridge between the two nuclei.
 
Imaging with RPA70-Turq and standard IF showed that the chromatin bridges accumulate RPA. The RPA binding suggested that nucleolytic attack is responsible for severing the bridges but attempts to identify nuclear nucleases involved in bridge breakage did not yield positive results.
 
An important clue about the fate of the dicentric chromosomes emerged from the observation that cells connected by chromatin bridges frequently suffer from transient nuclear envelope rupture during interphase (NERDI). NERDI correlated with diminished lamin staining on the chromatin bridges as well as the primary nuclear envelopes, a known cause of nuclear envelope rupture in cancer cell lines.
 
Given that the cells with chromatin bridges undergo NERDI, which exposes the nucleus to cytoplasmic factors, we asked whether the major cytoplasmic 3’ exonuclease, TREX1, contributes to bridge resolution. IF demonstrated TREX1 at the chromatin bridges and preliminary analysis of CRISPR-generated TREX1-/- cells suggested that TREX1 is required for the appearance of RPA on the chromatin. Furthermore, reduced TREX1 levels appeared to delay the resolution of the chromatin bridges.
 
The data indicate that dicentric chromosome resolution does not involve a simple single break event in anaphase or during cytokinesis. In stead the processing is more complex and likely involves extensive nucleolytic attack. The resulting fragmentation may underlie more complex genomic rearrangements, such as chromothripsis.
 
Coauthors: John Maciejowski, Nazario Bosco, Titia de Lange
Rockefeller University, New York, New York, United States

Mechanisms of Genome Inheritance during C. elegans Meiosis
Anne M. Villeneuve, PhD, Stanford University School of Medicine, Stanford, California, United States

Our long-term goal is to elucidate the mechanisms that govern the faithful inheritance of genomes during meiosis, the specialized cell division program by which diploid organisms generate haploid gametes. Chromosome inheritance during meiosis relies on the formation of double-strand DNA breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). Because the DSBs that serve as the initiating events of meiotic recombination pose a danger to genome integrity, the success of genome inheritance during meiosis requires cells to maintain a balance between the beneficial effects of COs and the potential harmful consequences of the process by which they are generated. Our goal is to understand the mechanisms that operate during meiosis to achieve this crucial balance. We are approaching this problem using the nematode C. elegans, a simple metazoan organism that is especially amenable to combining sophisticated cytological, genetic and genomic approaches in a single experimental system, and in which the events under study are particularly accessible. I will discuss evidence that meiosis operates as an integrated biological system in which multiple "engineering design features" collaborate to ensure a robust outcome. The features include: positive and negative feedback, self-organization / self-limiting properties, checkpoint/quality control mechanisms, enforcement and fail-safe mechanisms. I will focus on recent work using microscopic imaging strategies designed to elucidate the architecture and organization of DNA repair complexes at in vivo sites of meiotic recombination and to investigate the relationships between recombination events and meiosis-specific chromosome structures.
 
Coauthors: Divya Pattabiraman, MSc, Baptiste Roelens, PhD, Chloe Girard, PhD, Sreejith Ramakrishnan, PhD and Alexander Woglar, PhD
Stanford University School of Medicine, Stanford, California, United States

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