Kyle M. Miller

Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map

Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal; however, even knowledge of the stoichiometry, affinity and lifetime of every protein–protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein–protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.

Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining

DNA double-strand break (DSB) repair occurs within chromatin and can be modulated by chromatin-modifying enzymes. Here we identify the related human histone deacetylases HDAC1 and HDAC2 as two participants in the DNA-damage response. We show that acetylation of histone H3 Lys56 (H3K56) was regulated by HDAC1 and HDAC2 and that HDAC1 and HDAC2 were rapidly recruited to DNA-damage sites to promote hypoacetylation of H3K56. Furthermore, HDAC1- and 2-depleted cells were hypersensitive to DNA-damaging agents and showed sustained DNA-damage signaling, phenotypes that reflect defective DSB repair, particularly by nonhomologous end-joining (NHEJ). Collectively, these results show that HDAC1 and HDAC2 function in the DNA-damage response by promoting DSB repair and thus provide important insights into the radio-sensitizing effects of HDAC inhibitors that are being developed as cancer therapies.

Small-molecule–induced DNA damage identifies alternative DNA structures in human genes

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here, we show that the G-quadruplex interacting drug pyridostatin promoted growth arrest in human cancer cells via inducing replication- and transcription-dependent DNA damage. Chromatin immunoprecipitation sequence (ChIP-Seq) analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage, and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein levels and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.

Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells

Recognition and repair of damaged DNA occurs within the context of chromatin. The key protein components of chromatin are histones, whose post‐translational modifications control diverse chromatin functions. Here, we report our findings from a large‐scale screen for DNA‐damage‐responsive histone modifications in human cells. We have identified specific phosphorylations and acetylations on histone H3 that decrease in response to DNA damage. Significantly, we find that DNA‐damage‐induced changes in H3S10p, H3S28p and H3.3S31p are a consequence of cell‐cycle re‐positioning rather than DNA damage per se. In contrast, H3K9Ac and H3K56Ac, a mark previously uncharacterized in human cells, are rapidly and reversibly reduced in response to DNA damage. Finally, we show that the histone acetyl‐transferase GCN5/KAT2A acetylates H3K56 in vitro and in vivo. Collectively, our data indicate that though most histone modifications do not change appreciably after genotoxic stress, H3K9Ac and H3K56Ac are reduced in response to DNA damage in human cells.

Semi-conservative DNA replication through telomeres requires Taz1

Telomere replication is achieved through the combined action of the conventional DNA replication machinery and the reverse transcriptase, telomerase. Telomere-binding proteins have crucial roles in controlling telomerase activity; however, little is known about their role in controlling semi-conservative replication, which synthesizes the bulk of telomeric DNA. Telomere repeats in the fission yeast Schizosaccharomyces pombe are bound by Taz1, a regulator of diverse telomere functions. It is generally assumed that telomere-binding proteins impede replication fork progression. Here we show that, on the contrary, Taz1 is crucial for efficient replication fork progression through the telomere. Using two-dimensional gel electrophoresis, we find that loss of Taz1 leads to stalled replication forks at telomeres and internally placed telomere sequences, regardless of whether the telomeric G-rich strand is replicated by leading- or lagging-strand synthesis. In contrast, the Taz1-interacting protein Rap1 is dispensable for efficient telomeric fork progression. Upon loss of telomerase, taz1Δ telomeres are lost precipitously, suggesting that maintenance of taz1Δ telomere repeats cannot be sustained through semi-conservative replication. As the human telomere proteins TRF1 and TRF2 are Taz1 orthologues, we predict that one or both of the human TRFs may orchestrate fork passage through human telomeres. Stalled forks at dysfunctional human telomeres are likely to accelerate the genomic instability that drives tumorigenesis.

Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks

Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation–sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation–associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination

The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.

The telomere protein Taz1 is required to prevent and repair genomic DNA breaks

One fundamental function of telomeres is to prevent the ends of chromosomes from being sensed and treated as DNA damage. Here we present evidence for additional roles of telomeres in promoting proper chromosome segregation and DNA repair. We find that the fission yeast telomere protein Taz1p is required for cell cycle progression at 20 degrees C, a temperature at which taz1Delta cells exhibit a G(2)/M DNA damage checkpoint delay, chromosome missegregation, and DNA double-strand breaks (DSBs). Spindle assembly checkpoint components and a checkpoint-independent function of Rad3p are required for taz1Delta cells to survive at 20 degrees C. Disruption of topoisomerase II activity suppresses the cold sensitivity of taz1Delta cells, suggesting a scenario in which telomeric entanglement is the primary defect. Furthermore, hypersensitivity to treatments that induce DSBs suggests that Taz1p is involved in DSB repair. Our observations imply roles for Taz1p-containing telomeres in preventing and repairing DNA breaks throughout the genome.

Mammalian DNA repair: HATs and HDACs make their mark through histone acetylation

Genetic information is recorded in specific DNA sequences that must be protected to preserve normal cellular function. Genome maintenance pathways have evolved to sense and repair DNA damage. Importantly, deleterious mutations that occur from mis-repaired lesions can lead to diseases such as cancer. As eukaryotic DNA is bound by histone proteins and organized into chromatin, the true in vivo substrate of transcription, replication and DNA repair is chromatin. Almost 50 years ago, it was found that histones contained the post-translational modification (PTM), acetylation. With the cloning and identification of transcription associated histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that write and erase the histone acetylation mark respectively, it was realized that this histone modification could be dynamically regulated. Chromatin is subjected to numerous PTMs that regulate chromatin structure and function, including DNA repair. As different organisms contain different histone modifications, chromatin-associated proteins and chromatin states, it is likely that chromatin-templated processes such as DNA repair will exhibit organismal differences. This article focuses on the DNA damage response (DDR) in mammalian cells and how the concerted activities of HAT and HDAC enzymes, and their histone acetylation targets, specifically participate in DNA double-strand break (DSB) repair. Defects in DNA repair and chromatin pathways are observed in cancer, and these pathways represent cancer therapeutic targets. Therefore, understanding the relationship between DNA repair and histone acetylations is important for providing mechanistic details of DSB repair within chromatin that has the potential to be exploited in the clinic.