Sébastien Soubeyrand

Phosphorylation of Artemis following irradiation-induced DNA damage

Artemis is a DNA repair factor required for V(D)J recombination, repair of DNA damage induced by ionizing radiation (IR) or radiomimetic drugs, and the maintenance of genome integrity. During V(D)J recombination, Artemis participates in the resolution of hairpin‐sealed coding ends, a step crucial to the constitution of the gene encoding for the antigen receptor of lymphocytes. The precise role of Artemis in the repair of IR‐induced DNA damage remains to be elucidated. Here we show that Artemis is constitutively phosphorylated in cultured cells and undergoes additional phosphorylation events after irradiation. The IR‐induced phosphorylation is mainly, although not solely, dependent on Ataxia‐telangiectasia‐mutated kinase (ATM). The physiological role of these phosphorylation events remains unknown, as in vitro‐generated Artemis mutants, which present impaired IR‐induced phosphorylation, still display an activity sufficient to complement the V(D)J recombination defect and the increased radiosensibility of Artemis‐deficient cells. Thus, Artemis is an effector of DNA repair that can be phosphorylated by ATM, and possibly by DNA‐PKcs and ATR depending upon the type of DNA damage.

A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators

BackgroundMitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques.ResultsIn order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction.ConclusionsThe adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions.See Commentary: http://www.biomedcentral.com/1741-7007/8/99

Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins.

DNA-dependent protein kinase (DNA-PK) acts through an essential relationship with DNA to participate in the regulation of multiple cellular processes. Yet the role of DNA as a cofactor in kinase activity remains to be completely elucidated. For example, although DNA-PK activity appears to be required for the resolution of hairpin coding ends in variable diversity joining recombination, kinase activity remains to be demonstrated from hairpin ends or other DNA structures. In the present study we report that DNA-PK is strongly activated from hairpin ends and structured single-stranded DNA, but that the phosphorylation of many heterologous substrates is blocked efficiently by inactivation of the kinase through autophosphorylation. However, substrates that bound efficiently to single-stranded DNA such as p53 and replication protein A were efficiently phosphorylated by DNA-PK from structured DNA. DNA-PK also was found to be active toward heterologous substrates from hairpin ends on double-stranded DNA under conditions where autophosphorylation was minimized. These results suggest that the role of DNA-PK in resolving coding end hairpins is likely to be enzymatic rather than structural, expand understanding of how DNA-PK binding to structured DNA relates to enzyme activity, and suggest a mechanism for autoregulatory control of its kinase activity in the cell.

Functional Analysis of the TRIB1 Associated Locus Linked to Plasma Triglycerides and Coronary Artery Disease

Background The TRIB1 locus has been linked to hepatic triglyceride metabolism in mice and to plasma triglycerides and coronary artery disease in humans. The lipid‐associated single nucleotide polymorphisms (SNPs), identified by genome‐wide association studies, are located ≈30 kb downstream from TRIB1, suggesting complex regulatory effects on genes or pathways relevant to hepatic triglyceride metabolism. The goal of this study was to investigate the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits. Methods and Results Characterization of the risk locus reveals that it encompasses a gene, TRIB1‐associated locus (TRIBAL), composed of a well‐conserved promoter region and an alternatively spliced transcript. Bioinformatic analysis and resequencing identified a single SNP, rs2001844, within the promoter region that associates with increased plasma triglycerides and reduced high‐density lipoprotein cholesterol and coronary artery disease risk. Further, correction for triglycerides as a covariate indicated that the genome‐wide association studies association is largely dependent on triglycerides. In addition, we show that rs2001844 is an expression trait locus (eQTL) for TRIB1 expression in blood and alters TRIBAL promoter activity in a reporter assay model. The TRIBAL transcript has features typical of long noncoding RNAs, including poor sequence conservation. Modulation of TRIBAL expression had limited impact on either TRIB1 or lipid regulatory genes mRNA levels in human hepatocyte models. In contrast, TRIB1 knockdown markedly increased TRIBAL expression in HepG2 cells and primary human hepatocytes. Conclusions These studies demonstrate an interplay between a novel locus, TRIBAL, and TRIB1. TRIBAL is located in the genome‐wide association studies identified risk locus, responds to altered expression of TRIB1, harbors a risk SNP that is an eQTL for TRIB1 expression, and associates with plasma triglyceride concentrations.

Topoisomerase IIα-dependent induction of a persistent DNA damage response in response to transient etoposide exposure

Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25μM etoposide was highly toxic and initiated a double‐stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA‐PKcs to γH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and γH2AX response to correlate with the inability of the cell to correct topoIIα‐initiated DNA damage. γH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre‐treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIα‐dependent DNA defects within topologically open chromatin domains.

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease

Objective—A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-&bgr; pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease–associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. Approach and Results—By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D′, 0.97; r2, 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. Conclusions—The coronary artery disease–associated rs17293632C>T single nucleotide polymorphism represents a novel functional cis-acting element at the SMAD3 locus. The protective (T) allele of rs17293632 disrupts a consensus AP-1 binding site in a SMAD3 intron 1 enhancer, reduces enhancer activity and SMAD3 expression, altering human arterial smooth muscle cell proliferation.

Functional interaction between COL4A1/COL4A2 and SMAD3 risk loci for coronary artery disease

OBJECTIVE The COL4A1/COL4A2 region on chromosome 13q34 is a highly replicated locus for coronary artery disease (CAD). In the normal arterial wall, type IV collagen acts to inhibit smooth muscle cell proliferation. Its production is in part a function of TGFβ signaling, but the specific regulatory mechanisms, especially in humans, have not been defined. Our aim was to decipher TGFβ signaling components important in the regulation of COL4A1 and COL4A2 and determine whether these components showed genetic interaction with the COL4A1/COL4A2 locus for CAD association. METHODS AND RESULTS Experiments were performed in primary human aortic smooth muscle cells and HT1080 fibroblasts. Pharmacological inhibition of the TGFβ1 receptor and subsequent SMAD protein phosphorylation by treatment with an ALK5 inhibitor prevented the increase in COL4A1/COL4A2 mRNA (p < 0.001) and protein expression in response to TGFβ1 stimulation. In contrast, inhibition of the non-canonical TGFβ signaling pathways was without effect. siRNA mediated knockdown of SMAD3 and SMAD4 abolished the stimulatory effects of TGFβ1 on COL4A1/COL4A2 (p < 0.001) whereas SMAD2 knockdown had no effect. In luciferase reporter assays, neither SMAD3 overexpression nor TGFβ1 treatment altered COL4A1 or COL4A2 promoter activity, supportive of more complex regulation of type IV collagen gene expression by the TGFβ/SMAD3 signaling pathway. Epistasis analysis in 5 CAD case/control cohorts revealed that SMAD3 and COL4A1/COL4A2 display statistical interaction for CAD association. CONCLUSIONS These findings demonstrate that SMAD3 is a necessary factor for TGFβ-mediated stimulation of mRNA and protein expression of type IV collagen genes in human vascular smooth muscle cells. Epistasis analyses further supports the hypothesis that the SMAD3-dependent regulation of COL4A1/COL4A2 may be of functional significance for CAD pathogenesis.

ERK1/2 regulates hepatocyte Trib1 in response to mitochondrial dysfunction.

The TRIB1 locus (8q24.13) is a novel locus identified and replicated by several genome-wide association studies for associations with plasma triglycerides, apolipoprotein B and coronary artery disease. The TRIB1 protein product, tribbles-like protein 1 (Trib1), regulates MAPK activity. MAP kinases transduce a large variety of external signals, leading to a wide range of cellular responses, including growth, differentiation, inflammation and apoptosis. Importantly, Trib1 has been shown to regulate hepatic lipogenesis and very low density lipoprotein production. Despite the relevance of hepatocyte Trib1 to lipid metabolism and atherosclerosis, little is known about the mechanisms regulating Trib1 itself. Here, we identify the mitochondria axis as a regulator of Trib1. Treatment of HepG2 cells with a short pulse of a low oligomycin concentration led to a potent and prolonged increase in the Trib1 mRNA, an effect that was shared with other mitochondria stressors. HuH7 cells as well murine hepatocytes were also responsive albeit to a weaker extent. The upregulation appeared largely independent of reactive oxygen species generation or metabolic stress and was mainly under transcriptional control, with ERK1/2 playing an important regulating role in the process. While the presence of the Trib1 protein could be inferred, attempts to correlate the increased mRNA to changes in protein level were unsuccessful due to the lack of recognizable Trib1 signal. Our data enrich the current paradigm of Trib1 as an activator of the MAPK pathway by uncovering a role for MAPK in regulating Trib1.

TRIB1 Is Regulated Post-Transcriptionally by Proteasomal and Non-Proteasomal Pathways.

The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCPβ did not play a role in mediating TRIB1 instability although TRCPβ suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level.

Role of Tribbles Pseudokinase 1 (TRIB1) in human hepatocyte metabolism

Genome-wide association studies for plasma triglycerides and hepatic steatosis identified a risk locus on chromosome 8q24 close to the TRIB1 gene, encoding Tribbles Pseudokinase 1 (TRIB1). In previous studies conducted in murine models, hepatic over-expression of Trib1 was shown to increase fatty acid oxidation and decrease triglyceride synthesis whereas Trib1 knockdown mice exhibited hypertriglyceridemia. Here we have examined the impact of TRIB1 suppression in human and mouse hepatocytes. Examination of a panel of lipid regulator transcripts revealed species-specific effects, prompting us to focus on human models for the remainder of the study. Acute knockdown of TRIB1 in human primary hepatocytes resulted in decreased expression of MTTP and APOB, required for very low density lipoprotein (VLDL) assembly although particle secretion was not significantly affected. A parallel analysis performed in HepG2 revealed reduced MTTP, but not APOB, protein as a result of TRIB1 suppression. Global gene expression changes of human primary hepatocytes upon TRIB1 suppression were analyzed by clustering algorithms and found to be consistent with dysregulation of several pathways fundamental to liver function, including altered CEBPA and B transcript levels and impaired glucose handling. Indeed, TRIB1 expression in HepG2 cells was found to be inversely proportional to glucose concentration. Lastly TRIB1 downregulation in primary hepatocytes was associated with suppression of the HNF4A axis. In HepG2 cells, TRIB1 suppression resulted in reduced HNF4A protein levels while HNF4A suppression increased TRIB1 expression. Taken together these studies reveal an important role for TRIB1 in human hepatocyte biology.