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Purpose Inherited pathogenic variants in PALB2 are associated with increased risk of breast and pancreatic cancer. However, the functional and clinical relevance of many missense variants of uncertain significance (VUS) identified through clinical genetic testing is unclear. The ability of patient-derived germline missense VUS to disrupt PALB2 function was assessed to identify variants with potential clinical relevance. Methods The influence of 84 VUS on PALB2 function was evaluated using a cellular homology directed DNA repair (HDR) assay and VUS impacting activity were further characterized using secondary functional assays. Results Four (~5%) variants (p.L24S,c.71T>C; p.L35P,c.104T>C; pI944N,c.2831T>A; and p.L1070P,c.3209T>C) disrupted PALB2-mediated HDR activity. These variants conferred sensitivity to cisplatin and a poly(ADP-ribose) polymerase (PARP) inhibitor and reduced RAD51 foci formation in response to DNA damage. The p.L24S and p.L35P variants disrupted BRCA1–PALB2 protein complexes, p.I944N was associated with protein instability, and both p.I944N and p.L1070P mislocalized PALB2 to the cytoplasm. Conclusion These findings show that the HDR assay is an effective method for screening the influence of inherited variants on PALB2 function, that four missense variants impact PALB2 function and may influence cancer risk and response to therapy, and suggest that few inherited PALB2 missense variants disrupt PALB2 function in DNA repair.

Since its discovery, partner and localizer of breast cancer 2 (BRCA2) (PALB2) has emerged as a major tumor suppressor gene linked to breast cancer (BC), pancreatic cancer (PC), and ovarian cancer (OC) susceptibility. Its protein product plays a pivotal role in the maintenance of genome integrity. Here we discuss the first functional evaluation of a large set of PALB2 missense variants of uncertain significance (VUSs). Assessment of 136 VUSs interrogating a range of PALB2 biological functions resulted in the identification of 15 variants with consistent loss of function across different assays. All loss-of-function variants are located at the PALB2 coiled coil (CC) or at the WD40 domain, highlighting the importance of modular domains mechanistically involved in the DNA damage response (DDR) and pinpointing their roles in tumor suppression.

The PALB2 protein is essential to RAD51-mediated homologous recombination (HR) repair. Germline monoallelic PALB2 pathogenic variants confer significant risks for breast cancer. However, the majority of PALB2 variants remain classified as variants of unknown significance (VUS). We aim to functionally and mechanistically evaluate three novel PALB2 VUS. Patient-derived lymphoblastoid cell lines containing the VUS were analyzed for nuclear localization and foci formation of RAD51 as a measure of HR efficiency. To understand the mechanism underlying the HR deficiency, PALB2 nuclear localization was assessed using immunofluorescence studies. Among these VUS, c.3251C>T (p.Ser1084Leu) occurred in a patient with metastatic breast cancer while c.1054G>C (p.Glu352Gln) and c.1057A>G (p.Lys353Glu) were seen in patients with squamous cell carcinoma of skin and renal cell carcinoma respectively. Variant c.3251C>T was located within the WD40 domain which normally masked the nuclear export signal sequence responsible for nuclear delocalization of PALB2. Correspondingly, c.3251C>T displayed aberrant cytoplasmic localization of PALB2 which led to an impaired RAD51 nuclear localization and foci formation. On the other hand, both c.1054G>C and c.1057A>G showed intact HR functions and nuclear localization of PALB2, consistent with their locations within domains of no known function. Additionally, the prevalence of c.1054G>C was similar among healthy controls and patients with breast cancer (as seen in other studies), suggestive of its non-pathogenicity. In conclusion, our studies provided the functional evidence showing the deleterious effect of c.3251C>T, and non-deleterious effects of c.1054G>C and c.1057A>G. Using the ClinGen Pathogenicity calculator, c.3251C>T remains a VUS while c.1054G>C and c.1057A>G may be classified as likely benign variants.

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. While truncating variants in PALB2 are known to increase cancer risk, the interpretation of missense variants of uncertain significance (VUS) is in its infancy. Here we describe the development of a relatively fast and easy cDNA-based system for the semi high-throughput functional analysis of 48 VUS in human PALB2. By assessing the ability of PALB2 VUS to rescue the DNA repair and checkpoint defects in Palb2 knockout mouse embryonic stem (mES) cells, we identify various VUS in PALB2 that impair its function. Three VUS in the coiled-coil domain of PALB2 abrogate the interaction with BRCA1, whereas several VUS in the WD40 domain dramatically reduce protein stability. Thus, our functional assays identify damaging VUS in PALB2 that may increase cancer risk.

One typical mechanism to promote genomic instability, a hallmark of cancer, is to inactivate tumor suppressors, such as PALB2. It has recently been reported that mutations in PALB2 increase the risk of breast cancer by 8–9-fold by age 40 and the life time risk is ∼3–4-fold. To date, predicting the functional consequences of PALB2 mutations has been challenging as they lead to different cancer risks. Here, we performed a structure–function analysis of PALB2, using PALB2 truncated mutants (R170fs, L531fs, Q775X and W1038X), and uncovered a new mechanism by which cancer cells could drive genomic instability. Remarkably, the PALB2 W1038X mutant, harboring a mutation in its C-terminal domain, is still proficient in stimulating RAD51-mediated recombination in vitro, although it is unusually localized to the cytoplasm. After further investigation, we identified a hidden NES within the WD40 domain of PALB2 and found that the W1038X truncation leads to the exposure of this NES to CRM1, an export protein. This concept was also confirmed with another WD40-containing protein, RBBP4. Consequently, our studies reveal an unreported mechanism linking the nucleocytoplasmic translocation of PALB2 mutants to cancer formation.

APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions.

While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

Partner and Localizer of BRCA2 (PALB2) has emerged as an important and versatile player in genome integrity maintenance. Biallelic mutations in PALB2 cause Fanconi anemia (FA) subtype FA-N, whereas monoallelic mutations predispose to breast, and pancreatic familial cancers. Herein, we review recent developments in our understanding of the mechanisms of regulation of the tumor suppressor PALB2 and its functional domains. Regulation of PALB2 functions in DNA damage response and repair occurs on multiple levels, including homodimerization, phosphorylation, and ubiquitylation. With a molecular emphasis, we present PALB2-associated cancer mutations and their detailed analysis by functional assays.

SUMMARY All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance.

Summary ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR.

Summary The interplay between homologous DNA recombination and mitotic progression is poorly understood. The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) are key enzymes for DNA double-strand break repair. In our search for specific functions of the various RAD51 paralogs, we found that inhibition of XRCC3 elicits checkpoint defects, while inhibition of RAD51B or RAD51C induces G2/M cell cycle arrest in HeLa cells. Using live-cell microscopy we show that in XRCC3-knockdown cells the spindle assembly checkpoint persists and there is a higher frequency of chromosome misalignments, anaphase bridges, and aneuploidy. We observed centrosome defects in the absence of XRCC3. While RAD51B and RAD51C act early in homologous recombination, XRCC3 functions jointly with GEN1 later in the pathway at the stage of Holliday junction resolution. Our data demonstrate that Holliday junction resolution has critical functions for preventing aberrant mitosis and aneuploidy in mitotic cells.

2-Ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) is a novel anticancer agent that arrests cell cycle in S-phase and causes DNA replication stress leading to the phosphorylation of H2AX into γ-H2AX. First, using the M21, HT29, HT-1080 and HeLa cell lines, we confirmed that S-phase cell cycle arrest and γ-H2AX foci induction by SFOM-0046 is a general mechanism occurring in diverse cancer cell lines. In addition to γ-H2AX, SFOM-0046 activates preferentially ATR-Chk1 in M21 and HT29 cells while both ATR-Chk1 and ATM-Chk2 pathways are activated in HCT116 cells. Co-localization of SFOM-0046-induced 53BP1 foci with γ-H2AX foci validates that the DNA damage generated corresponds to double-strand-breaks (DSBs). Consistent with an S-phase arrest, SFOM-0046 treatment induces RAD51 foci formation but not DNA-PKcs foci, confirming that homologous recombination is the major DSB repair pathway targeted by the drug. Furthermore, using isogenic HCT116 p53+/+ and HCT116 p53−/− cells, we showed that p53 plays a key role in the survival mechanism to SFOM-0046. Finally, SFOM-0046 exhibits a dose-dependent antitumor activity on human fibrosarcoma HT-1080 tumours grafted onto chick chorioallantoic membranes without showing embryo toxicity even at high doses. Altogether, our results highlight SFOM-0046 as a very promising drug that induces a replication stress response.

Uncovering the biological role of nuclear receptor peroxisome proliferator-activated receptors (PPARs) has greatly advanced our knowledge of the transcriptional control of glucose and energy metabolism. As such, pharmacological activation of PPARγ has emerged as an efficient approach for treating metabolic disorders with the current use of thiazolidinediones to improve insulin resistance in diabetic patients. The recent identification of growth hormone releasing peptides (GHRP) as potent inducers of PPARγ through activation of the scavenger receptor CD36 has defined a novel alternative to regulate essential aspects of lipid and energy metabolism. Recent advances on the emerging role of CD36 and GHRP hexarelin in regulating PPARγ downstream actions with benefits on atherosclerosis, hepatic cholesterol biosynthesis and fat mitochondrial biogenesis are summarized here. The response of PPARγ coactivator PGC-1 is also discussed in these effects. The identification of the GHRP-CD36-PPARγ pathway in controlling various tissue metabolic functions provides an interesting option for metabolic disorders.

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.

Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARgamma activation by hexarelin, and phosphorylation of PPARgamma was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARgamma activation function-1 activity. However, the activation of PPARgamma by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)alpha, suggesting a differential regulation of PPARgamma-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARgamma agonist, the occupancy of the CD36 promoter by PPARgamma was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRalpha promoter was strongly occupied by PPARgamma in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARgamma and LXRalpha target genes in peritoneal macrophages. The response was strongly impaired in PPARgamma(+/-) macrophages, indicating that PPARgamma was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARgamma and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.

One typical mechanism to promote genomic instability, a hallmark of cancer, is to inactivate tumor suppressors, such as PALB2. It has recently been reported that mutations in PALB2 increase the risk of breast cancer by 8-9 fold. PALB2 was identified BRCA2 interacting protein, essential for BRCA2 anchorage to nuclear structures and for its function in double-strand break repair. Inherited mutations in PALB2 are associated with a predisposition for ovarian, breast and pancreatic cancers. The basis of the tumorigenic potential of PALB2 is thought to be related to functions in homologous recombination. Therefore, the regulation of PALB2 during the DNA damage response and the effect of cancer-causing mutation is of high interest. Two mechanisms of regulation of PALB2 will be presented. The first mechanism regulates PALB2 localization to DNA damage sites in S-phase. To date, predicting the functional consequences of PALB2 mutations has been challenging as they lead to different aggressive phenotypes. Here, we performed a structure-function analysis of PALB2 using PALB2 truncated mutants (R170fs, L531fs, Q775X and W1038X), and uncovered a second PALB2 regulation mechanism by which cancer cells could drive genomic instability. We will present these regulatory mechanisms and synthetic lethal strategies to kill PALB2 deficient cells harbouring such mutations using PARP inhibitors. These strategies also apply to APRIN, A BRCA2 interactor, which also promote double-strand break repair by homologous recombination. Citation Format: Anthony M. Couturier, Rémi Buisson, Joris Pauty, Amélie Rodrigue, Marie-Christine Caron, Yan Coulombe, Niraj Joshi, Lee Zou, Jean-Yves Masson. Mechanisms of regulation and synthetic lethal strategies against PALB2 and APRIN, two DNA double-strand break repair proteins [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B18.

Cancer is a constant threat to humans since one out of three individuals will develop cancer during their lifetime. It has become increasingly clear that tumor formation can be triggered by mutations in enzymes involved in the surveillance of genome integrity, such as BRCA1, BRCA2 and PALB2. BRCA1, BRCA2 and PALB2 are essential players in double–strand break repair by homologous recombination and have been associated with a heightened lifetime risk for ovarian cancer development. Cancer cells with BRCA1/2 and PALB2 deficiency are extremely sensitive to inhibitors of the DNA repair protein PARP, which have recently emerged as promising anti–cancer drugs. However, mutations in BRCA1/2 and PALB2 account only for around 15–20 % of ovarian cancers overall. Developing new strategies to specifically target ovarian cancer is still presenting a major challenge. During homologous recombination, PALB2 links BRCA1 and BRCA2 to promote RAD51 filament formation at DNA double–strand breaks repair. PALB2 interacts directly with BRCA1 via its N–terminal coiled–coil domain and with BRCA2 via its C–terminal WD40 domain. After DNA damage, PALB2–BRCA1 interaction is enhanced to promote PALB2, BRCA2 and RAD51 localization to DNA double–strand breaks. However, how DNA damage promotes the interaction between PALB2 and BRCA1 is still not understood. In this study, we found that the phosphatidylinositol 3–kinase–like proteins kinase ATR is essential to promote PALB2 and RAD51 to DNA double–strand breaks by enhancing the interaction between PALB2 and BRCA1. We identified two functions of ATR important to enhance PALB2–BRCA1 interaction. First, ATR directly phosphorylates PALB2 on serine 59 after DNA damage. Secondly after DNA double–strand breaks resection, ATR down–regulates CDKs activity leading to the decrease of serine 64 phosphorylation on PALB2. Together, these dual events lead to a direct enhancement of the interaction between PALB2 and BRCA1. Furthermore, we generated PALB2 mutants mimicking the active and inactive state of PALB2. We showed that PALB2 phospho–mutants that recapitulated the active state of PALB2 are able to bypass the absence of ATR activity in cells while mutants that mimic the inactive state of PALB2 showed a defect even in presence of active ATR. These results explain for the first time why ATR is essential to promote DNA double–strand break repair by homologous recombination. My results suggest that that ovarian cancer cells that do not carry BRCA1/2 or PALB2 mutations may be rendered “BRCA–like” by treatment with ATR inhibitor, making them susceptible to treatments with PARPi and others DNA damaging drugs. Citation Format: Rémi Buisson, Niraj Joshi, Chu Kwen Ho, Amélie Rodrigue, Tzeh Keong Foo, Emilie Hardy, Wilhelm Haas, Bing Xia, Jean–Yves Masson and Lee Zou. ATR–DEPENDENT REGULATION OF THE OVARIAN CANCER PROTEIN PALB2 AT DNA DOUBLE–STRAND BREAKS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-049.

One typical mechanism to promote genomic instability, a hallmark of cancer, is to inactivate tumor suppressors, such as PALB2. It has recently been reported that mutations in PALB2 increase the risk of breast cancer by 8-9 fold. PALB2 was identified BRCA2 interacting protein, essential for BRCA2 anchorage to nuclear structures and for its function in double-strand break repair. Inherited mutations in PALB2 are associated with a predisposition for ovarian, breast and pancreatic cancers. The basis of the tumorigenic potential of PALB2 is thought to be related to functions in homologous recombination. Therefore, the regulation of PALB2 during the DNA damage response and the effect of cancer-causing mutation is of high interest. Two mechanisms of regulation of PALB2 will be presented. The first mechanism regulates PALB2 localization to DNA damage sites in S-phase through ATR-mediated phosphorylation. We have found specific phosphorylated residues in PALB2 that promote BRCA1-PALB2 interaction after DNA damage. To date, predicting the functional consequences of PALB2 mutations has been challenging as they lead to different aggressive phenotypes. We performed a structure-function analysis of PALB2 using PALB2 truncated mutants (R170fs, L531fs, Q775X and W1038X), and uncovered a second PALB2 regulation mechanism. Our data explains the tumorigenesis associated with some PALB2 mutations, a concept that also applies to other WD40-containing proteins. This abstract is also being presented as Poster B11. Citation Format: Anthony Couturier, Rémi Buisson, Joris Pauty, Amélie Rodrigue, Niraj Joshi, Marie-Christine Caron, Yan Coulombe, Lee Zou, Jean-Yves Masson. Mechanisms of regulation of the tumor suppressor PALB2 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR01.

PALB2 [partner and localizer of BRCA2 (breast cancer early-onset 1)] has emerged as a key player in the maintenance of genome integrity. Biallelic mutations in PALB2 cause FA (Fanconi's anaemia) subtype FA-N, a devastating inherited disorder marked by developmental abnormalities, bone marrow failure and childhood cancer susceptibility, whereas monoallelic mutations predispose to breast, ovarian and pancreatic cancer. The tumour suppressor role of PALB2 has been intimately linked to its ability to promote HR (homologous recombination)-mediated repair of DNA double-strand breaks. Because PALB2 lies at the crossroads between FA, HR and cancer susceptibility, understanding its function has become the primary focus of several studies. The present review discusses a current synthesis of the contribution of PALB2 to these pathways. We also provide a molecular description of FA- or cancer-associated PALB2 mutations.

The scavenger receptor CD36 plays a central role in lipid metabolism by promoting macrophage cholesterol efflux with the potential to reduce atherosclerotic lesions. However, the effect of CD36 on de novo cholesterol synthesis is not known. Here, we describe the cellular mechanism by which CD36 activation induces cholesterol depletion in HepG2 cells. Using the CD36 ligand hexarelin, we found a rapid phosphorylation of HMG-CoA reductase Ser-872 in treated cells, resulting in inactivation of the rate-limiting enzyme in sterol synthesis. Degradation of HMG-CoA reductase by the ubiquitin-proteasome pathway was also enhanced by hexarelin, through an increased recruitment of the anchor proteins insulin-induced gene (Insig)-1 and Insig-2. Genes encoding key enzymes involved in cholesterol synthesis and under the control of transcription factor sterol regulatory element-binding protein (SREBP)-2 remained unresponsive to sterol depletion, due to retention of the SREBP-2 escort protein Scap by Insig-1/2. Insig1 and Insig2 gene expression was also increased through activation of nuclear receptor peroxisome-proliferator activating receptor γ (PPARγ) by CD36, which lifted the inhibitory effect of PPARγ1 Ser-84 phosphorylation. Recruitment of coactivator peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) to activated AMPKα was also promoted, resulting in PGC-1α transcriptional activation through Sirt1-mediated deacetylation, increased recruitment of PPARγ, and up-regulation of Insig-1/2, revealing a regulatory role of CD36 on PGC-1α signaling. Our data identify CD36 as a novel regulator of HMG-CoA reductase function and Insig-1/2 expression, 2 critical steps regulating cholesterol synthesis in hepatocytes.

Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.

Background The PARP family member poly(ADP-ribose) polymerase 3 (PARP3) is structurally related to the well characterized PARP1 that orchestrates cellular responses to DNA strand breaks and cell death by the synthesis of poly(ADP-ribose). In contrast to PARP1 and PARP2, the functions of PARP3 are undefined. Here, we reveal critical functions for PARP3 during vertebrate development. Principal Findings We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins. We demonstrate that PARP3 gene occupancy in the human neuroblastoma cell line SK-N-SH occurs preferentially with developmental genes regulating cell fate specification, tissue patterning, craniofacial development and neurogenesis. Addressing the significance of this association during zebrafish development, we show that morpholino oligonucleotide-directed inhibition of parp3 expression in zebrafish impairs the expression of the neural crest cell specifier sox9a and of dlx3b/dlx4b, the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud. Conclusion Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.

International audience; Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Investigating the metabolic functions of the nuclear receptor peroxisome proliferator-activated receptorγ(PPARγ) has been extremely rewarding over the past years. Uncovering the biologic roles of PPARγand its mechanism of action has greatly advanced our understanding of the transcriptional control of lipid and glucose metabolism, and compounds such as thiazolidinediones which directly regulate PPARγhave proven to exhibit potent insulin-sensitizer effects in the treatment of diabetes. We review here recent advances on the emerging role of growth hormone releasing peptides in regulating PPARγthrough interaction with scavenger receptor CD36 and ghrelin GHS-R1a receptor. With the impact that these peptides exert on the metabolic pathways involved in lipid metabolism and energy homeostasis, it is hoped that the development of novel approaches in the regulation of PPAR functions will bring additional therapeutic possibilities to face problems related to metabolic diseases.

Whereas the uptake of oxidized lipoproteins by scavenger receptor CD36 in macrophages has been associated with foam cell formation and atherogenesis, little is known about the role of CD36 in regulating lipid metabolism in adipocytes. Here we report that treatment of 3T3-L1 adipocytes with hexarelin, a GH-releasing peptide that interacts with CD36, resulted in a depletion of intracellular lipid content with no significant change in CD36 expression. Microarray analysis revealed an increased pattern in several genes involved in fatty acid mobilization toward the mitochondrial oxidative phosphorylation process in response to hexarelin. Interestingly, many of these up-regulated genes are known targets of peroxisomal proliferator-activated receptor (PPAR)-gamma, such as FATP, CPT-1, and F(1)-ATPase, suggesting that adipocyte response to hexarelin may involve PPARgamma activation. Expression studies also indicate an increase in thermogenic markers PPARgamma coactivator 1alpha and uncoupling protein-1, which are normally expressed in brown adipocytes. Electron microscopy of hexarelin-treated 3T3-L1 adipocytes showed an intense and highly organized cristae formation that spans the entire width of mitochondria, compared with untreated cells, and cytochrome c oxidase activity was enhanced by hexarelin, two features characteristic of highly oxidative tissues. A similar mitochondrial phenotype was detected in epididymal white fat of mice treated with hexarelin, along with an increased expression of thermogenic markers that was lost in treated CD36-null mice, suggesting that the ability of hexarelin to promote a brown fat-like phenotype also occurs in vivo and is dependent on CD36. These results provide a potential role for CD36 to impact the overall metabolic activity of fat usage and mitochondrial biogenesis in adipocytes.