Person: Kim, Hyun-Min
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Kim
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Hyun-Min
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Kim, Hyun-Min
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Publication Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes(Genetics Society of America, 2015) Norris, Adam D.; Kim, Hyun-Min; Colaiacovo, Monica; Calarco, John AUse of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4–5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments.Publication NatB Domain-Containing CRA-1 Antagonizes Hydrolase ACER-1 Linking Acetyl-CoA Metabolism to the Initiation of Recombination during C. elegans Meiosis(Public Library of Science, 2015) Gao, Jinmin; Kim, Hyun-Min; Elia, Andrew; Elledge, Stephen; Colaiacovo, MonicaThe formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.Publication Interplay between Structure-Specific Endonucleases for Crossover Control during Caenorhabditis elegans Meiosis(Public Library of Science, 2013) Saito, Takamune T.; Lui, Doris Y.; Kim, Hyun-Min; Meyer, Katherine; Colaiacovo, MonicaThe number and distribution of crossover events are tightly regulated at prophase of meiosis I. The resolution of Holliday junctions by structure-specific endonucleases, including MUS-81, SLX-1, XPF-1 and GEN-1, is one of the main mechanisms proposed for crossover formation. However, how these nucleases coordinately resolve Holliday junctions is still unclear. Here we identify both the functional overlap and differences between these four nucleases regarding their roles in crossover formation and control in the Caenorhabditis elegans germline. We show that MUS-81, XPF-1 and SLX-1, but not GEN-1, can bind to HIM-18/SLX4, a key scaffold for nucleases. Analysis of synthetic mitotic defects revealed that MUS-81 and SLX-1, but not XPF-1 and GEN-1, have overlapping roles with the Bloom syndrome helicase ortholog, HIM-6, supporting their in vivo roles in processing recombination intermediates. Taking advantage of the ease of genetic analysis and high-resolution imaging afforded by C. elegans, we examined crossover designation, frequency, distribution and chromosomal morphology in single, double, triple and quadruple mutants of the structure-specific endonucleases. This revealed that XPF-1 functions redundantly with MUS-81 and SLX-1 in executing crossover formation during meiotic double-strand break repair. Analysis of crossover distribution revealed that SLX-1 is required for crossover suppression at the center region of the autosomes. Finally, analysis of chromosome morphology in oocytes at late meiosis I stages uncovered that SLX-1 and XPF-1 promote meiotic chromosomal stability by preventing formation of chromosomal abnormalities. We propose a model in which coordinate action between structure-specific nucleases at different chromosome domains, namely MUS-81, SLX-1 and XPF-1 at the arms and SLX-1 at the center region, exerts positive and negative regulatory roles, respectively, for crossover control during C. elegans meiosis.Publication REST and Stress Resistance in Ageing and Alzheimer’s Disease(Nature Publishing Group, 2014) Lu, Tao; Aron, Liviu; Zullo, Joseph; Pan, Ying; Kim, Haeyoung; Chen, Yiwen; Yang, Tun-Hsiang; Kim, Hyun-Min; Drake, Derek; Liu, Xiaole; Bennett, David A.; Colaiacovo, Monica; Yankner, BruceHuman neurons are functional over an entire lifetime, yet the mechanisms that preserve function and protect against neurodegeneration during ageing are unknown. Here we show that induction of the repressor element 1-silencing transcription factor (REST; also known as neuron-restrictive silencer factor, NRSF) is a universal feature of normal ageing in human cortical and hippocampal neurons. REST is lost, however, in mild cognitive impairment and Alzheimer’s disease. Chromatin immunoprecipitation with deep sequencing and expression analysis show that REST represses genes that promote cell death and Alzheimer’s disease pathology, and induces the expression of stress response genes. Moreover, REST potently protects neurons from oxidative stress and amyloid β-protein toxicity, and conditional deletion of REST in the mouse brain leads to age-related neurodegeneration. A functional orthologue of REST, Caenorhabditis elegans SPR-4, also protects against oxidative stress and amyloid β-protein toxicity. During normal ageing, REST is induced in part by cell non-autonomous Wnt signalling. However, in Alzheimer’s disease, frontotemporal dementia and dementia with Lewy bodies, REST is lost from the nucleus and appears in autophagosomes together with pathological misfolded proteins. Finally, REST levels during ageing are closely correlated with cognitive preservation and longevity. Thus, the activation state of REST may distinguish neuroprotection from neurodegeneration in the ageing brain.Publication ZTF-8 Interacts with the 9-1-1 Complex and Is Required for DNA Damage Response and Double-Strand Break Repair in the C. elegans Germline(Public Library of Science, 2014) Kim, Hyun-Min; Colaiacovo, MonicaGermline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.