Benjamin Barré

A high-definition view of functional genetic variation from natural yeast genomes

The question of how genetic variation in a population influences phenotypic variation and evolution is of major importance in modern biology. Yet much is still unknown about the relative functional importance of different forms of genome variation and how they are shaped by evolutionary processes. Here we address these questions by population level sequencing of 42 strains from the budding yeast Saccharomyces cerevisiae and its closest relative S. paradoxus. We find that genome content variation, in the form of presence or absence as well as copy number of genetic material, is higher within S. cerevisiae than within S. paradoxus, despite genetic distances as measured in single-nucleotide polymorphisms being vastly smaller within the former species. This genome content variation, as well as loss-of-function variation in the form of premature stop codons and frameshifting indels, is heavily enriched in the subtelomeres, strongly reinforcing the relevance of these regions to functional evolution. Genes affected by these likely functional forms of variation are enriched for functions mediating interaction with the external environment (sugar transport and metabolism, flocculation, metal transport, and metabolism). Our results and analyses provide a comprehensive view of genomic diversity in budding yeast and expose surprising and pronounced differences between the variation within S. cerevisiae and that within S. paradoxus. We also believe that the sequence data and de novo assemblies will constitute a useful resource for further evolutionary and population genomics studies.

A Quantitative Proteomic Approach of the Different Stages of Colorectal Cancer Establishes OLFM4 as a New Nonmetastatic Tumor Marker

Expression profiles represent new molecular tools that are useful to characterize the successive steps of tumor progression and the prediction of recurrence or chemotherapy response. In this study, we have used quantitative proteomic analysis to compare different stages of colorectal cancer. A combination of laser microdissection, OFFGEL separation, iTRAQ labeling, and MALDI-TOF/TOF MS was used to explore the proteome of 28 colorectal cancer tissues. Two software packages were used for identification and quantification of differentially expressed proteins: Protein Pilot and iQuantitator. Based on ∼1,190,702 MS/MS spectra, a total of 3138 proteins were identified, which represents the largest database of colorectal cancer realized to date and demonstrates the value of our quantitative proteomic approach. In this way, individual protein expression and variation have been identified for each patient and for each colorectal dysplasia and cancer stage (stages I–IV). A total of 555 proteins presenting a significant fold change were quantified in the different stages, and this differential expression correlated with immunohistochemistry results reported in the Human Protein Atlas database. To identify a candidate biomarker of the early stages of colorectal cancer, we focused our study on secreted proteins. In this way, we identified olfactomedin-4, which was overexpressed in adenomas and in early stages of colorectal tumors. This early stage overexpression was confirmed by immunohistochemistry in 126 paraffin-embedded tissues. Our results also indicate that OLFM4 is regulated by the Ras-NF-κB2 pathway, one of the main oncogenic pathways deregulated in colorectal tumors.

The STAT3 Transcription Factor Is a Target for the Myc and Riboblastoma Proteins on the Cdc25A Promoter

The STAT3 (signal transducer and activator of transcription) transcription factor functions as down-stream effector of growth factor signaling. Whereas STAT3 activation is transient in normal cells, constitutively activated forms of the transcription factor have been detected in several cancer cell lines and primary tumors. Through the up-regulation of cell cycle and survival genes, STAT3 plays important roles in cell growth, anti-apoptosis, and cell transformation yet the molecular basis for this behavior is poorly understood. In this study, we show that STAT3 and its transcriptional cofactors are recruited to the promoter of the Cdc25A gene to activate its expression. Using chromatin immunoprecipitations, we observed that Myc is recruited to this promoter following STAT3 DNA binding. Moreover, small interfering RNA-mediated knockdown of Myc specifically inhibits the STAT3-mediated activation of Cdc25A. Reduction in Myc protein level results in defective recruitment of the CREB-binding protein, Cdk9, and RNA polymerase complexes, indicating that Myc is necessary for STAT3 transcription. Surprisingly, the association of STAT3 with the Cdc25A promoter does not necessarily lead to transcriptional induction because this protein also functions as a transcriptional repressor of the Cdc25A gene. Following hydrogen peroxide stimulation, STAT3 forms a repressor complex with the retinoblastoma (Rb) tumor suppressor to occupy the Cdc25A promoter and block its induction. In coimmunoprecipitations and ChIP experiments, Rb was found to associate with STAT3 on DNA and we provide evidence that Rb binds directly to the transcription factor. Thus, we propose that Myc and STAT3 cooperate to induce the expression of Cdc25A and that their transcriptional activity is normally regulated by the Rb tumor suppressor gene.

The cell cycle inhibitor p21waf1 binds to the myc and cdc25A promoters upon DNA damage and induces transcriptional repression.

In addition to its function as a cyclin-dependent kinase (cdk) inhibitor, p21waf1 fulfills additional roles involved in DNA replication and transcriptional regulation that could also contribute to cell cycle arrest. In this study, we have shown that p21waf1 functions as a transcriptional repressor of the myc and cdc25A genes. Ectopic expression of the cell cycle inhibitor down-modulates myc and cdc25A transcription but has no effect on cdk4 levels. Using chromatin immunoprecipitation, we found that p21waf1 is recruited to the promoters of these two genes together with the STAT3 and E2F1 transcription factors. Its presence on DNA is associated with an inhibition of the recruitment of the p300 histone acetylase and with a down-regulation of histone H4 acetylation. The same effect was also observed following DNA damage because topoisomerase inhibitors such as sn38 or doxorubicin also induce the association of p21waf1 with DNA. Following transcriptional repression of the myc and cdc25A genes, cells were arrested in the fraction with 4 N DNA content. By contrast, the expression of these two genes remains elevated in the absence of the cell cycle inhibitor, and p21waf1–/– cells re-replicate their DNA and become polyploid. In light of these results, we propose that p21waf1 simultaneously targets cdk and transcriptional regulators to prevent the expression of oncogenic pathways upon DNA damage.

The cdk5 kinase regulates the STAT3 transcription factor to prevent DNA damage Upon topoisomerase I inhibition

The STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to growth factor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes involved in cell-cycle progression. Despite its importance in cancer cells, the molecular mechanisms by which this protein is regulated in response to DNA damage remain to be characterized. In this study, we show that STAT3 is activated in response to topoisomerase I inhibition. Following treatment, STAT3 is phosphorylated on its C-terminal serine 727 residue but not on its tyrosine 705 site. We also show that topoisomerase I inhibition induced the up-regulation of the cdk5 kinase, a protein initially described in neuronal stress responses. In co-immunoprecipitations, cdk5 was found to associate with STAT3, and pulldown experiments indicated that it associates with the C-terminal activation domain of STAT3 upon DNA damage. Importantly, the cdk5-STAT3 pathway reduced DNA damage in response to topoisomerase I inhibition through the up-regulation of Eme1, an endonuclease involved in DNA repair. ChIP experiments indicated that STAT3 can be found associated with the Eme1 promoter when phosphorylated only on its serine 727 residue and not on tyrosine 705. We therefore propose that the cdk5-STAT3 oncogenic pathway plays an important role in the expression of DNA repair genes and that these proteins could be used as predictive markers of tumors that will fail to respond to chemotherapy.

How should we define STAT3 as an oncogene and as a potential target for therapy

Aberrant activation of the STAT3 transcription factor has been reported in a large group of tumors and a strong biological basis now defines this protein as an oncogenic driver. Consequently, STAT3 is considered to be a promising target in the field of cancer therapy. For its inhibition to result in a successful therapeutic approach, the definition of a target tumor population identified by specific and detectable alterations is critical. The canonical activation model of STAT3 relies on a constitutive phosphorylation on its 705 tyrosine site, resulting in its dimerization, nuclear translocation, and the consequent activation of cancer genes. Therefore, it is expected that tumors expressing this phosphorylated form are addicted to STAT3 and will be sensitive to existing drugs which are targeting this dimeric form. However, recent results have shown that STAT3 can function as an oncogene in the absence of this tyrosine phosphorylation. This indicates that different forms of the transcription factor also play an important role in tumor growth and chemotherapy resistance. This complicates the definition of STAT3 as an oncogene and as a potential prognosis and predictive biomarker. The obligation to target a defined tumor type implies that future clinical trials should use a precise definition of STAT3 activation. This will allow tumors addicted to this oncogene to be identified correctly, leading to a strong rationale for patient stratification.

Regulation of activity and function of the p52 NF-κB subunit following DNA damage

We have previously shown that after DNA-damage, the p52 NF-kB subunit can function cooperatively with the p53 tumor suppressor to both repress and induce Skp2 expression. However, the wider role and activation of p52 after DNA-damage has not been determined. Activation of NF-kB in response to DNA break inducers can be mediated by ATM (ataxia telangiectasia mutated)-dependent phosphorylation of NEMO (NF-kB essential modulator), resulting in IKKβ mediated induction of the classical NF-kB pathway, leading to the induction of RelA(p65)/p50 dimers. Here, we show that DNA damage also induces p100 (NF-kB2) processing to generate active p52. We further demonstrate that p52 generation is dependent not only on IKKα but also on atypical activation by NEMO/ATM. Moreover, we identify a post-DNA damage, positive feedback loop of p52 activation through induction of NF-kB2 gene expression, involving both the classical and alternative NF-kB pathways. Gene expression and chromatin immunoprecipitation analyses indicated DNA damage induced p52 dimer recruitment on multiple, p53 dependent and independent, target genes associated with promoting cell cycle arrest and cell death. These results demonstrate an important role for the alternative, p52 NF-kB pathway after DNA-damage distinct from its functions as a regulator of adaptive immunity.

Escape from p21-mediated Oncogene-induced Senescence Leads to Cell Dedifferentiation and Dependence on Anti-apoptotic Bcl-xL and MCL1 Proteins *

Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16INK4, HT29 cells enter senescence, up-regulate p21WAF1, and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAFv600E in LS174T cells. We also observed that p21WAF1 prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21WAF1 binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4–5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21WAF1 down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21WAF1 escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24low/CD44high phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21WAF1 are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.

Genome evolution across 1,011 Saccharomyces cerevisiae isolates

Large-scale population genomic surveys are essential to explore the phenotypic diversity of natural populations. Here we report the whole-genome sequencing and phenotyping of 1,011 Saccharomyces cerevisiae isolates, which together provide an accurate evolutionary picture of the genomic variants that shape the species-wide phenotypic landscape of this yeast. Genomic analyses support a single ‘out-of-China’ origin for this species, followed by several independent domestication events. Although domesticated isolates exhibit high variation in ploidy, aneuploidy and genome content, genome evolution in wild isolates is mainly driven by the accumulation of single nucleotide polymorphisms. A common feature is the extensive loss of heterozygosity, which represents an essential source of inter-individual variation in this mainly asexual species. Most of the single nucleotide polymorphisms, including experimentally identified functional polymorphisms, are present at very low frequencies. The largest numbers of variants identified by genome-wide association are copy-number changes, which have a greater phenotypic effect than do single nucleotide polymorphisms. This resource will guide future population genomics and genotype–phenotype studies in this classic model system.Whole-genome sequencing of 1,011 natural isolates of the yeast Saccharomyces cerevisiae reveals its evolutionary history, including a single out-of-China origin and multiple domestication events, and provides a framework for genotype–phenotype studies in this model organism.

STAT3 as a new autophagy regulator

Signal transducers and activators of transcription 3 (STAT3) proteins are cytoplasmic transcription factors that translocate into the nucleus to induce transcription following growth factor or cytokine stimulation. Besides their normal functions, these proteins play an important role in cancer cells through the abnormal activation of cell cycle progression and the deregulation of survival and senescence pathways. New data obtained from the laboratory of Guido Kroemer identifies STAT3 as a new autophagy regulator. In the cytoplasm, in the absence of conventional phosphorylation on the tyrosine 705 residue, STAT3 interacts with the PKR kinase to inhibit eIF2A phosphorylation and so reduce autophagic pathways. This new and nonconventional function of STAT3 has an important role in normal cells but we suggest that it might also affect cancer cells and the response to chemotherapy treatment.