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Bcl-2 associated factor 1 (Bclaf1) is a nuclear protein that was originally identified in a screen of proteins that interact with the adenoviral bcl-2 homolog E1B19K. Overexpression of Bclaf1 was shown to result in apoptosis and transcriptional repression that was reversible in the presence of Bcl-2 or Bcl-xL. Furthermore, antiapoptotic members, but not proapoptotic members of the Bcl-2 protein family, were shown to interact with Bclaf1 and prevent its localization to the nucleus. Bclaf1 has also recently been identified as a binding partner for Emerin, a nuclear membrane protein that is mutated in X-linked recessive Emery–Dreifuss muscular dystrophy. To ascertain the in vivo function of Bclaf1, we have generated mice that carry a targeted mutation of the bclaf1 locus. In this study, we show that Bclaf1 is required for proper spatial and temporal organization of smooth muscle lineage during the saccular stage of lung development. We also show that Bclaf1 is dispensable for thymocyte development but is essential for peripheral T-cell homeostasis. Despite its postulated role as a proapoptotic protein, Bclaf1-deficient cells did not show any defect in cell death linked to development or after exposure to various apoptotic stimuli. Our findings show a critical role for Bclaf1 in developmental processes independent of apoptosis.Cell Death and Differentiation (2009) 16, 331–339; doi:10.1038/cdd.2008.167; published online 14 November 2008 [ABSTRACT FROM AUTHOR]

In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAF Δβ3-αC oncoproteins usually lack five amino acids in the β3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAF Δβ3-αC oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAF ΔLNVTAP>F and two novel mutants, BRAF delinsFS and BRAF ΔLNVT>F , and compare them with other BRAF Δβ3-αC oncoproteins. We show that BRAF Δβ3-αC oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAF Δβ3-αC oncoproteins, e.g., BRAF ΔLNVTAP>F , increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAF Δβ3-αC mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.

The SARS-CoV-2 pandemic highlighted the need for rapid, collaborative, and population-centric research to define health impact, develop health care policies and establish reliable diagnostic and surveillance tests. Critical for these objectives were in-depth clinical data collected in standardized fashion and large numbers of various types of human samples prior and post-viral encounter. As the pandemic evolved with the emergence of new variants of concern (VOCs), access to samples and data from infected and vaccinated individuals were needed to monitor immune durability, the possibility of increased transmissibility and virulence, and vaccine protection against new and emerging VOCs. Therefore, essential to the pandemic response is a strong laboratory and data research component, supported by effective biobanking and data sharing. Critically important to the speed of the research response is the rapid access to biobanked samples. To address critical challenges brought to light by the pandemic, the Coronavirus Variants Rapid Response Network (CoVaRR-Net), funded by the Canadian Institutes of Health Research, was established to coordinate research efforts to provide rapid evidence-based responses to emerging VOCs. The purpose of this paper is to introduce the CoVaRR-Net Biobank and define its contribution to pandemic preparedness., (© Association of Medical Microbiology and Infectious Disease Canada (AMMI Canada), 2022.)

Purpose: To investigate the robustness and longevity of SARS-CoV-2 immune responses conferred by natural infection and vaccination among priority populations such as immunocompromised individuals and people with post-acute sequelae of COVID-19 in a prospective cohort study (Stop the Spread Ottawa-SSO) in adults living in the Ottawa region. In this paper, we describe the study design, ongoing data collection and baseline characteristics of participants., Participants: Since October 2020, participants who tested positive for COVID-19 (convalescents) or at high risk of exposure to the virus (under surveillance) have provided monthly blood and saliva samples over a 10-month period. As of 2 November 2021, 1026 adults had completed the baseline survey and 976 had attended baseline bloodwork. 300 participants will continue to provide bimonthly blood samples for 24 additional months (ie, total follow-up of 34 months)., Findings to Date: The median age of the baseline sample was 44 (IQR 23, range: 18-79) and just over two-thirds (n=688; 67.1%) were female. 255 participants (24.9%) had a history of COVID-19 infection confirmed by PCR and/or serology. Over 600 participants (60.0%) work in high-risk occupations (eg, healthcare, teaching and transportation). 108 participants (10.5%) reported immunocompromising conditions or treatments at baseline (eg, cancer, HIV, other immune deficiency, and/or use of immunosuppressants)., Future Plans: SSO continues to yield rich research potential, given the collection of pre-vaccine baseline data and samples from the majority of participants, recruitment of diverse subgroups of interest, and a high level of participant retention and compliance with monthly sampling. The 24-month study extension will maximise opportunities to track SARS-CoV-2 immunity and vaccine efficacy, detect and characterise emerging variants, and compare subgroup humoral and cellular response robustness and persistence., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Intratumoral heterogeneity is a critical frontier in understanding how the tumor microenvironment (TME) propels malignant progression. Here, we deconvolute the human pancreatic TME through large-scale integration of histology-guided regional multiOMICs with clinical data and patient-derived preclinical models. We discover "subTMEs," histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response. "Reactive" subTMEs rich in complex but functionally coordinated fibroblast communities were immune hot and inhabited by aggressive tumor cell phenotypes. The matrix-rich "deserted" subTMEs harbored fewer activated fibroblasts and tumor-suppressive features yet were markedly chemoprotective and enriched upon chemotherapy. SubTMEs originated in fibroblast differentiation trajectories, and transitory states were notable both in single-cell transcriptomics and in situ. The intratumoral co-occurrence of subTMEs produced patient-specific phenotypic and computationally predictable heterogeneity tightly linked to malignant biology. Therefore, heterogeneity within the plentiful, notorious pancreatic TME is not random but marks fundamental tissue organizational units., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide. There is an unmet need to develop novel clinically relevant models of NSCLC to accelerate identification of drug targets and our understanding of the disease., Experimental Design: Thirty surgically resected NSCLC primary patient tissue and 35 previously established patient-derived xenograft (PDX) models were processed for organoid culture establishment. Organoids were histologically and molecularly characterized by cytology and histology, exome sequencing, and RNA-sequencing analysis. Tumorigenicity was assessed through subcutaneous injection of organoids in NOD/SCID mice. Organoids were subjected to drug testing using EGFR, FGFR, and MEK-targeted therapies., Results: We have identified cell culture conditions favoring the establishment of short-term and long-term expansion of NSCLC organoids derived from primary lung patient and PDX tumor tissue. The NSCLC organoids recapitulated the histology of the patient and PDX tumor. They also retained tumorigenicity, as evidenced by cytologic features of malignancy, xenograft formation, preservation of mutations, copy number aberrations, and gene expression profiles between the organoid and matched parental tumor tissue by whole-exome and RNA sequencing. NSCLC organoid models also preserved the sensitivity of the matched parental tumor to targeted therapeutics, and could be used to validate or discover biomarker-drug combinations., Conclusions: Our panel of NSCLC organoids closely recapitulates the genomics and biology of patient tumors, and is a potential platform for drug testing and biomarker validation., (©2019 American Association for Cancer Research.)

TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an aryl hydrocarbon receptor (AHR) target gene that functions as part of a negative feedback loop to repress AHR activity. Tiparp -/- mice exhibit increased sensitivity to the toxicological effects of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), including lethal wasting syndrome. However, it is not known whether Tiparp -/- mice also exhibit increased sensitivity to other AHR ligands. In this study, we treated male Tiparp -/- or wild type (WT) mice with a single injection of 100 mg/kg 3-methylcholanthrene (3MC). Consistent with TIPARP's role as a repressor of AHR signaling, 3MC-treated Tiparp -/- mice exhibited increased hepatic Cyp1a1 and Cyp1b1 levels compared with WT mice. No 3MC-treated Tiparp -/- mice survived beyond day 16 and the mice exhibited chylous ascites characterized by an accumulation of fluid in the peritoneal cavity. All WT mice survived the 30-day treatment and showed no signs of fluid accumulation. Treated Tiparp -/- mice also exhibited a transient and mild hepatotoxicity with inflammation. 3MC-treated WT, but not Tiparp -/- mice, developed mild hepatic steatosis. Lipid deposits accumulated on the surface of the liver and other abdominal organs in the 3MC- Tiparp -/- mice. Our study reveals that Tiparp -/- mice have increased sensitivity to 3MC-induced liver toxicity, but unlike with TCDD, lethality is due to chylous ascites rather than wasting syndrome.

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo- p -dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33 ITPLKTCFK 41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR., (© 2018 The Author(s).)

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparpfl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparpfl/flCreAlb) and whole-body (Tiparpfl/flCreCMV; TiparpEx3-/-) Tiparp null mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice given a single injection of 10 μg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparpfl/flCreAlb and TiparpEx3-/- mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparpfl/flCreAlb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.

Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRβ activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and in Tiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/β peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRβ co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRβ. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced in Tiparp(-/-)mice compared with Tiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1., (© 2016 Authors; published by Portland Press Limited.)

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp(-/-) mice given a single injection of 100 μg/kg dioxin did not survive beyond day 5; all Tiparp(+/+) mice survived the 30-day treatment. Dioxin-treated Tiparp(-/-) mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP(-/-) mouse embryonic fibroblasts (MEFs) and AHRR(-/-) MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP(-/-) MEFs, AHRR(-/-) MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR(-/-) MEFs or AHRRΔ8, the active isoform of AHRR, in TiPARP(-/-) MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRRΔ8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRRΔ8_Δ1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRRΔ8_Δ1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRRΔ8_Δ1-49 nor GFP-AHRRΔ8_Δ1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1(-/-)) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1(-/-) MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity in the Ogg1(-/-) MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1(-/-) MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Neurodevelopmental defects are observed in the hereditary disorder Cockayne syndrome (CS). The gene most frequently mutated in CS, Cockayne Syndrome B (CSB), is required for the repair of bulky DNA adducts in transcribed genes during transcription-coupled nucleotide excision repair. CSB also plays a role in chromatin remodeling and mitochondrial function. The role of CSB in neural development is poorly understood. Here we report that the abundance of neural progenitors is normal in Csb(-/-) mice and the frequency of apoptotic cells in the neurogenic niche of the adult subependymal zone is similar in Csb(-/-) and wild type mice. Both embryonic and adult Csb(-/-) neural precursors exhibited defective self-renewal in the neurosphere assay. In Csb(-/-) neural precursors, self-renewal progressively decreased in serially passaged neurospheres. The data also indicate that Csb and the nucleotide excision repair protein Xpa preserve embryonic neural stem cell self-renewal after UV DNA damage. Although Csb(-/-) neural precursors do not exhibit altered neuronal lineage commitment after low-dose UV (1J/m(2)) in vitro, neurons differentiated in vitro from Csb(-/-) neural precursors that had been irradiated with 1J/m(2) UV exhibited defective neurite outgrowth. These findings identify a function for Csb in neural precursors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the PARP family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study, we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T-47D breast cancer and HuH-7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 messenger RNA (mRNA) expression levels and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not aryl hydrocarbon receptor nuclear translocator. Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc-finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Overexpression of Tiparp enhanced, whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was enhanced in Tiparp(-/-) mouse embryonic fibroblasts compared with wildtype controls. Our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop in AHR signalling.

Objectives: To determine any discordance between first and second brain death examinations and investigate the quality of brain death determination in Canadian PICUs., Design: Multicenter retrospective chart review., Setting: Four Canadian PICUs., Patients: All deaths from 1999 to 2003 were screened. Patients included were 36 wks corrected gestation to 17 yrs old and had at least one brain death examination documented in the chart., Interventions: None., Measurements and Main Results: Medical records were reviewed to determine demographics, results of the brain death examinations, ancillary tests, and time intervals between injury event, fixed dilated pupils, first brain death examination, second brain death examination, and terminal event. Discordance between brain death examinations was defined as the medical conclusion documented in the chart as brain death followed by no brain death. Prespecified subgroups were age <1 yr vs. ≥ 1 yr and organ donor vs. nonorgan donor. Mann-Whitney-Wilcoxon and unpaired t tests compared time intervals between subgroups; p value ≤ 0.05 was considered significant. Of those screened, 135 of 907 (15%) met the study eligibility criteria and 110 of 135 (81%) had at least two brain death examinations. The prevalence of discordance between brain death examinations was 1 of 110 (0.91%) (95% confidence interval <0.01%-5.5%). In those who had two apnea tests, the prevalence of discordance between brain death examinations was 1 of 63 (1.6%) (95% confidence interval <0.01%-9.3%). Twenty-five (19%) patients had only one brain death examination, and one of these became an organ donor without ancillary testing. Twenty-four (18%) patients did not have an apnea test. Of the 48 (36%) who had only one apnea test, 16 became organ donors without any ancillary test. Patients <1 yr old had a longer time interval between the first and second brain death examinations than those >1 yr old., Conclusions: Some brain death diagnoses were not based on two examinations, and some did not include an apnea test. In patients who had two brain death examinations, discordant results were uncommon.

Background: DNA double-strand breaks (DSBs) caused by ionizing radiation or by the stalling of DNA replication forks are among the most deleterious forms of DNA damage. The ability of cells to recognize and repair DSBs requires post-translational modifications to histones and other proteins that facilitate access to lesions in compacted chromatin, however our understanding of these processes remains incomplete. UHRF1 is an E3 ubiquitin ligase that has previously been linked to events that regulate chromatin remodeling and epigenetic maintenance. Previous studies have demonstrated that loss of UHRF1 increases the sensitivity of cells to DNA damage however the role of UHRF1 in this response is unclear., Results: We demonstrate that UHRF1 plays a critical role for facilitating the response to DSB damage caused by gamma-irradiation. UHRF1-depleted cells exhibit increased sensitivity to gamma-irradiation, suggesting a compromised cellular response to DSBs. UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (gammaH2AX) in response to gamma-irradiation compared to control cells. We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells., Conclusions: Our findings indicate a critical role for UHRF1 in maintenance of chromosome integrity and an optimal response to DSB damage.

Hexavalent chromium (Cr[VI]) is a toxic environmental contaminant that is capable of producing a broad spectrum of DNA damage. The ability of Cr[VI] to induce mutagenesis and neoplastic transformation has been attributed to its genotoxic action, however our understanding of molecular mechanisms involved in the repair of Cr[VI]-induced DNA damage remains incomplete. Here, we report that Mus81, an enzyme that participates with Eme1 in the resolution of replication fork damage caused by certain lesions, is involved in the repair of Cr[VI]-induced DNA damage. Mus81-deficient cells were found to be more susceptible to Cr[VI]-induced proliferation arrest and more sensitive to the long-term cytotoxic effects of Cr[VI] than isogenic wild-type cells. Following Cr[VI] exposure, Mus81-deficient cells displayed a lag in the disappearance of Rad51 foci, exhibited elevated replication-associated gamma-H2AX and showed an increased incidence of chromosomal instability compared to wild-type cells. Our findings support a role for Mus81 in the resolution of replication-associated DNA damage associated with this genotoxic agent, by converting Cr[VI]-DNA lesions into a form more amenable for homologous recombination.

Mus81 (methyl methansulfonate UV sensitive clone 81) and Eme1 (essential meiotic endonuclease 1, also known as MMS4) form a heterodimeric endonuclease that is critical for genomic stability and the response to DNA crosslink damage and replication blockade. However, relatively little is known as to how this endonuclease is regulated following DNA damage. Here, we report mammalian Eme1 interacts with Np95, an E3 ubiquitin ligase that participates in chromatin modification, replication-linked epigenetic maintenance and the DNA damage response. Np95 and Eme1 co-localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. Taken together, these findings link Mus81-Eme1 with the replication-associated chromatin modifier functions of Np95 in the cellular response to DNA damage.

Mus81 plays an integral role in the maintenance of genome stability and DNA repair in mammalian cells. Deficiency of Mus81 in human and mouse cells results in hypersensitivity to interstrand cross-linking (ICL) agents and elevated levels of genomic instability. Furthermore, Mus81-mutant mice are susceptible to spontaneous lymphomas. The role of cellular checkpoints in mediating the phenotypes observed in Mus81-deficient cells and mice is currently unknown. In this study, we have observed increased activation of p53 in Mus81(-/-) cells in response to ICL-induced DNA damage. In addition, p53 inactivation completely rescued the ICL hypersensitivity of Mus81(-/-) cells, signifying p53 is essential for the elimination of ICL-damaged cells in the absence of Mus81. Confirming that p53 acts as a critical checkpoint for the Mus81 repair pathway, a synergistic increase of spontaneous and ICL-induced genomic instability was observed in Mus81(-/-)p53(-/-) cells. To clarify the genetic interactions of Mus81 and p53 in tumor suppression, we monitored Mus81(-/-)p53(-/-) and control mice for the development of spontaneous tumors. Significantly, we show that loss of even a single allele of Mus81 drastically modifies the tumor spectrum of p53-mutant mice and increases their predisposition to developing sarcomas. Our results reveal a key role for p53 in mediating the response to spontaneous and ICL-induced DNA damage that occurs in the absence of Mus81. Furthermore, our data show that loss of Mus81, in addition to p53, is a key step in sarcoma development.

The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.

Disruption of Brca1 results in cellular demise or tumorigenesis depending on cellular context. Inactivation of p53 contributes to Brca1-associated tumor susceptibility. However the activation of p53-dependent checkpoint/apoptotic signaling in the absence of Brca1 is poorly understood. Here, we show that Chk2 inactivation is partially equivalent to p53 inactivation, in that Chk2 deficiency facilitates the development, survival, and proliferation of Brca1-deficient T cells at the expense of genomic integrity. Brca1 deficiency was found to result in Chk2 phosphorylation and the Chk2-dependent accumulation and activation of p53. Furthermore, inactivation of Chk2 and Brca1 was cooperative in breast cancer. Our findings identify a critical role for Chk2 as a component of the DNA damage-signaling pathway activated in response to Brca1 deficiency.

Defects in death receptor-mediated apoptosis have been linked to cancer and autoimmune disease in humans. The in vivo role of caspase 8, a component of this pathway, has eluded analysis in postnatal tissues because of the lack of an appropriate animal model. Targeted disruption of caspase 8 is lethal in utero. We generated mice with a targeted caspase 8 mutation that is restricted to the T-cell lineage. Despite normal thymocyte development in the absence of caspase 8, we observed a marked decrease in the number of peripheral T-cells and impaired T-cell response ex vivo to activation stimuli. caspase 8 ablation protected thymocytes and activated T-cells from CD95 ligand but not anti-CD3-induced apoptosis, or apoptosis activated by agents that are known to act through the mitochondria. caspase 8 mutant mice were unable to mount an immune response to viral infection, indicating that caspase 8 deletion in T-cells leads to immunodeficiency. These findings identify an essential, cell-stage-specific role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. This is consistent with the recent identification of caspase 8 mutations in human immunodeficiency.