Induction of \u3cem\u3eIL19\u3c/em\u3e Expression through JNK and cGAS-STING Modulates DNA Damage–Induced Cytokine Production

Cytokine production is a critical component of cell-extrinsic responses to DNA damage and cellular senescence. Here, we demonstrated that expression of the gene encoding interleukin-19 (IL-19) was enhanced by DNA damage through pathways mediated by c-Jun amino-terminal kinase (JNK) and cGAS-STING and that IL19 expression was required for the subsequent production of the cytokines IL-1, IL-6, and IL-8. IL19 expression was stimulated by diverse cellular stresses, including inhibition of the DNA replication checkpoint kinase ATR (ataxia telangiectasia and Rad3-related protein), oncogene expression, replicative exhaustion, oxidative stress, and DNA double-strand breaks. Unlike the production of IL-6 and IL-8, IL19 expression was not affected by abrogation of signaling by the IL-1 receptor (IL-1R) or the mitogen-activated protein kinase p38. Instead, the DNA damage–induced production of IL-1, IL-6, and IL-8 was substantially reduced by suppression of IL19 expression. The signaling pathways required to stimulate IL19 expression selectively depended on the type of DNA-damaging agent. Reactive oxygen species and the ASK1-JNK pathway were critical for responses to ionizing radiation (IR), whereas the cGAS-STING pathway stimulated IL19 expression in response to either IR or ATR inhibition. Whereas induction of IL1, IL6, and IL8 by IR depended on IL19 expression, the cGAS-STING–dependent induction of the immune checkpoint gene PDL1 after IR and ATR inhibition was independent of IL19. Together, these results suggest that IL-19 production by diverse pathways forms a distinct cytokine regulatory arm of the response to DNA damage

A Realistic Roadmap to Formation Flying Space Interferometry

The ultimate astronomical observatory would be a formation flying space interferometer, combining sensitivity and stability with high angular resolution. The smallSat revolution offers a new and maturing prototyping platform for space interferometry and we put forward a realistic plan for achieving first stellar fringes in space by 2030

Mechanisms of Common Fragile Site Instability and Cancer.

An increase in DNA damage is an “enabling characteristic” in tumorigenesis, and normally occurs at loci called common fragile sites (CFSs), or by the loss of genes involved in the DNA damage response pathway. CFSs are a normal part of the human genome, and are exceptionally prone to DNA damage under conditions of replication stress, such as treatment with low concentrations of the DNA polymerase inhibitor aphidicolin. While much has been learned about the cellular responses to DNA damage at CFSs, less is known about what makes these sites inherently unstable. To determine if the sequence of CFSs is causal to their instability, BACs containing CFS sequence were stably transfected into HCT116 cells, and cell clones with BACs integrated at ectopic non-fragile loci were isolated. Cell clones containing integrated CFS BACs showed a significant increase in aphidicolin-induced DNA damage at the integration site, compared to control BACs. This is the first direct evidence in human cells that introduction of CFS sequences into ectopic non-fragile loci is sufficient to cause CFS-like instability. These data support the hypothesis that sequences at CFSs are inherently unstable, and are a major factor in the formation of replication stress induced gaps and breaks at CFSs. A primary responder to DNA damage at CFSs, the protein kinase Ataxia-telangiectasia and rad3-related (ATR) is responsible for sensing and signaling DNA damage associated with stalled replication forks. The role of ATR and the maintenance of damage at CFSs, in tumorigenesis is not well understood due to the lethality of complete loss of ATR and is best studied in vivo. To overcome this limitation, we created a viable mouse model with a hypomorphic Atr mutation and an estimated 66-82% reduction in Atr protein levels. These mice exhibited increased APH-induced DNA damage and checkpoint abnormalities indicative of Atr deficiency. Creation of a mouse model with hypomorphic Atr expression will serve as a valuable reagent to study the biological effects of Atr deficiency in vivo.Ph.D.Human GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/63666/1/ryanrag_1.pd

Replication stress induces genome-wide copy number changes in human cells that resemble polymorphic and pathogenic variants

Copy number variants (CNVs) are an important component of genomic variation in humans and other mammals. Similar de novo deletions and duplications, or copy number changes (CNCs), are now known to be a major cause of genetic and developmental disorders and to arise somatically in many cancers. A major mechanism leading to both CNVs and disease-associated CNCs is meiotic unequal crossing over, or nonallelic homologous recombination (NAHR), mediated by flanking repeated sequences or segmental duplications. Others appear to involve nonhomologous end joining (NHEJ) or aberrant replication suggesting a mitotic cell origin. Here we show that aphidicolin-induced replication stress in normal human cells leads to a high frequency of CNCs of tens to thousands of kilobases across the human genome that closely resemble CNVs and disease-associated CNCs. Most deletion and duplication breakpoint junctions were characterized by short (<6 bp) microhomologies, consistent with the hypothesis that these rearrangements were formed by NHEJ or a replication-coupled process, such as template switching. This is a previously unrecognized consequence of replication stress and suggests that replication fork stalling and subsequent error-prone repair are important mechanisms in the formation of CNVs and pathogenic CNCs in humans

Differential roles for DNA polymerases eta, zeta, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross-links

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Polη), REV1, and zeta (Polζ) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polζ are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polζ facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Polη, whereas RAD18 plus Polζ, REV1, and Polζ are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links. Copyright © 2010, American Society for Microbiology. All Rights Reserved

Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR

Oncogenic Ras and p53 loss-of-function mutations are common in many advanced sporadic malignancies and together predict a limited responsiveness to conventional chemotherapy. Notably, studies in cultured cells have indicated that each of these genetic alterations creates a selective sensitivity to ataxia telangiectasia and Rad3-related (ATR) pathway inhibition. Here, we describe a genetic system to conditionally reduce ATR expression to 10% of normal levels in adult mice to compare the impact of this suppression on normal tissues and cancers in vivo. Hypomorphic suppression of ATR minimally affected normal bone marrow and intestinal homeostasis, indicating that this level of ATR expression was sufficient for highly proliferative adult tissues. In contrast, hypomorphic ATR reduction potently inhibited the growth of both p53-deficient fibrosarcomas expressing H-rasG12V and acute myeloid leukemias (AMLs) driven by MLL-ENL and N-rasG12D. Notably, DNA damage increased in a greater-than-additive fashion upon combining ATR suppression with oncogenic stress (H-rasG12V, K-rasG12D, or c-Myc overexpression), indicating that this cooperative genome-destabilizing interaction may contribute to tumor selectivity in vivo. This toxic interaction between ATR suppression and oncogenic stress occurred without regard to p53 status. These studies define a level of ATR pathway inhibition in which the growth of malignancies harboring oncogenic mutations can be suppressed with minimal impact on normal tissue homeostasis, highlighting ATR inhibition as a promising therapeutic strategy