Diauxic Inhibition: Jacques Monod's Ignored Work.

Diauxie is at the origin of research that led Jacques Monod (1910-1976), François Jacob (1920-2013), and André Lwoff (1902-1994) to win the Nobel Prize in Physiology or Medicine in 1965 for their description of the first genetic regulatory model. Diauxie is a term coined by Jacques Monod in 1941 in his doctoral dissertation that refers to microbial growth in two phases. In this article, we first examine Monod's thesis to demonstrate how and why Monod interpreted diauxie as a phenomenon of enzyme inhibition or suppression of adaptive enzymes. We also briefly investigate prior enzyme suppression studies, before Monod's work, which indicate that he is the first person to observe diauxic growth. Second, we analyse Monod's post-thesis publications throughout his scientific career, revealing that diauxic inhibition was a significant part of Monod's scientific activities and greatly fascinated Monod until the end of his life. Paradoxically, Monod's work and interest on diauxic inhibition are still neglected in historical recounts, focused mostly on Monod's enzymatic adaptation studies. Indeed, we uncovered a statement by Monod's colleague, Lwoff, who transformed a quotation from Monod by replacing the word phenomenon with enzymatic adaptation, which we believe has influenced historians. Finally, we offer hypotheses to explain why Lwoff altered Monod's statement.

Thermodynamically stable and genetically unstable G-quadruplexes are depleted in genomes across species.

G-quadruplexes play various roles in multiple biological processes, which can be positive when a G4 is involved in the regulation of gene expression or detrimental when the folding of a stable G4 impairs DNA replication promoting genome instability. This duality interrogates the significance of their presence within genomes. To address the potential biased evolution of G4 motifs, we analyzed their occurrence, features and polymorphisms in a large spectrum of species. We found extreme bias of the short-looped G4 motifs, which are the most thermodynamically stable in vitro and thus carry the highest folding potential in vivo. In the human genome, there is an over-representation of single-nucleotide-loop G4 motifs (G4-L1), which are highly conserved among humans and show a striking excess of the thermodynamically least stable G4-L1A (G3AG3AG3AG3) sequences. Functional assays in yeast showed that G4-L1A caused the lowest levels of both spontaneous and G4-ligand-induced instability. Analyses across 600 species revealed the depletion of the most stable G4-L1C/T quadruplexes in most genomes in favor of G4-L1A in vertebrates or G4-L1G in other eukaryotes. We discuss how these trends might be the result of species-specific mutagenic processes associated to a negative selection against the most stable motifs, thus neutralizing their detrimental effects on genome stability while preserving positive G4-associated biological roles.

Nuclear factor I X is a recurrent target for HPV16 insertions in anal carcinomas.

Anal carcinomas (AC) are associated with human papillomavirus (HPV) DNA sequences, but little is known about the physical state of the viral genome in carcinoma cells. To define the integration status and gene(s) targeted by viral insertions in AC, tumor DNAs extracted from 35 tumor specimen samples in patients with HPV16-associated invasive carcinoma were analyzed using the detection of integrated papillomavirus sequences-PCR approach. The genomic status at integration sites was assessed using comparative genomic hybridization-array assay and gene expression using reverse transcription quantitative PCR (RT-qPCR). HPV16 DNA was found integrated in 25/35 (71%) cases and the integration locus could be determined at the molecular level in 19 cases (29 total integration loci). HPV DNA was inserted on different chromosomes, but 5 cases harbored viral sequences at 19p13.2, within the nuclear factor I X (NFIX) locus. Viral DNA mapped between the most distal and the two proximal alternatively expressed exons of this gene in three cases (CA21, CA04, and CA35) and upstream of this gene (663 kb and 2.3 Mb) in the others. CGH arrays showed genomic gains/amplifications at the NFIX region, associated with HPV within the gene and RT-qPCR, revealed NFIX mRNA overexpression. Other genes targeted by integration were IL20RB, RPS6KA2, MSRA1, PIP5K1B, SLX4IP, CECR1, BCAR3, ATF6, CSNK1G1, APBA2, AGK, ILF3, PVT1, TRMT1, RAD51B, FASN, CCDC57, DSG3, and ZNF563. We identified recurrent targeting of NFIX by HPV16 insertion in anal carcinomas, supporting a role for this gene in oncogenesis, as reported for non-HPV tumors.

Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma.

Specific human papillomavirus genotypes are associated with most ano-genital carcinomas and a large subset of oro-pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus-associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus-16 or human papillomavirus-18-associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro-pharynx. As negative controls, 18 serum samples from women with human papillomavirus-16-associated high-grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at -80°C (27 cases) or at -20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus-16 E7 and human papillomavirus-18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at -80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus-associated invasive cancers even at sub-clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus-associated high grade cervical intraepithelial neoplasia.

Mechanistic signatures of HPV insertions in cervical carcinomas.

To identify new personal biomarkers for the improved diagnosis, prognosis and biological follow-up of human papillomavirus (HPV)-associated carcinomas, we developed a generic and comprehensive Capture-HPV method followed by Next Generation Sequencing (NGS). Starting from biopsies or circulating DNA samples, this Capture-NGS approach rapidly identifies the HPV genotype, HPV status (integrated, episomal or absence), the viral-host DNA junctions and the associated genome rearrangements. This analysis of 72 cervical carcinomas identified five HPV signatures. The first two signatures contain two hybrid chromosomal-HPV junctions whose orientations are co-linear (2J-COL) or non-linear (2J-NL), revealing two modes of viral integration associated with chromosomal deletion or amplification events, respectively. The third and fourth signatures exhibit 3-12 hybrid junctions, either clustered in one locus (MJ-CL) or scattered at distinct loci (MJ-SC) while the fifth signature consists of episomal HPV genomes (EPI). Cross analyses between the HPV signatures and the clinical and virological data reveal unexpected biased representation with respect to the HPV genotype, patient age and disease outcome, suggesting functional relevance(s) of this new classification. Overall, our findings establish a facile and comprehensive rational approach for the molecular detection of any HPV-associated carcinoma and definitive personalised sequence information to develop sensitive and specific biomarkers for each patient.

Genetic profiles of cervical tumors by high-throughput sequencing for personalized medical care.

Cancer treatment is facing major evolution since the advent of targeted therapies. Building genetic profiles could predict sensitivity or resistance to these therapies and highlight disease-specific abnormalities, supporting personalized patient care. In the context of biomedical research and clinical diagnosis, our laboratory has developed an oncogenic panel comprised of 226 genes and a dedicated bioinformatic pipeline to explore somatic mutations in cervical carcinomas, using high-throughput sequencing. Twenty-nine tumors were sequenced for exons within 226 genes. The automated pipeline used includes a database and a filtration system dedicated to identifying mutations of interest and excluding false positive and germline mutations. One-hundred and seventy-six total mutational events were found among the 29 tumors. Our cervical tumor mutational landscape shows that most mutations are found in PIK3CA (E545K, E542K) and KRAS (G12D, G13D) and others in FBXW7 (R465C, R505G, R479Q). Mutations have also been found in ALK (V1149L, A1266T) and EGFR (T259M). These results showed that 48% of patients display at least one deleterious mutation in genes that have been already targeted by the Food and Drug Administration approved therapies. Considering deleterious mutations, 59% of patients could be eligible for clinical trials. Sequencing hundreds of genes in a clinical context has become feasible, in terms of time and cost. In the near future, such an analysis could be a part of a battery of examinations along the diagnosis and treatment of cancer, helping to detect sensitivity or resistance to targeted therapies and allow advancements towards personalized oncology.

Lsd1 and lsd2 control programmed replication fork pauses and imprinting in fission yeast.

In the fission yeast Schizosaccharomyces pombe, a chromosomal imprinting event controls the asymmetric pattern of mating-type switching. The orientation of DNA replication at the mating-type locus is instrumental in this process. However, the factors leading to imprinting are not fully identified and the mechanism is poorly understood. Here, we show that the replication fork pause at the mat1 locus (MPS1), essential for imprint formation, depends on the lysine-specific demethylase Lsd1. We demonstrate that either Lsd1 or Lsd2 amine oxidase activity is required for these processes, working upstream of the imprinting factors Swi1 and Swi3 (homologs of mammalian Timeless and Tipin, respectively). We also show that the Lsd1/2 complex controls the replication fork terminators, within the rDNA repeats. These findings reveal a role for the Lsd1/2 demethylases in controlling polar replication fork progression, imprint formation, and subsequent asymmetric cell divisions.

Checkpoint-dependent and -independent roles of Swi3 in replication fork recovery and sister chromatid cohesion in fission yeast.

Multiple genome maintenance processes are coordinated at the replication fork to preserve genomic integrity. How eukaryotic cells accomplish such a coordination is unknown. Swi1 and Swi3 form the replication fork protection complex and are involved in various processes including stabilization of replication forks, activation of the Cds1 checkpoint kinase and establishment of sister chromatid cohesion in fission yeast. However, the mechanisms by which the Swi1-Swi3 complex achieves and coordinates these tasks are not well understood. Here, we describe the identification of separation-of-function mutants of Swi3, aimed at dissecting the molecular pathways that require Swi1-Swi3. Unlike swi3 deletion mutants, the separation-of-function mutants were not sensitive to agents that stall replication forks. However, they were highly sensitive to camptothecin that induces replication fork breakage. In addition, these mutants were defective in replication fork regeneration and sister chromatid cohesion. Interestingly, unlike swi3-deleted cell, the separation-of-functions mutants were proficient in the activation of the replication checkpoint, but their fork regeneration defects were more severe than those of checkpoint mutants including cds1Δ, chk1Δ and rad3Δ. These results suggest that, while Swi3 mediates full activation of the replication checkpoint in response to stalled replication forks, Swi3 activates a checkpoint-independent pathway to facilitate recovery of collapsed replication forks and the establishment of sister chromatid cohesion. Thus, our separation-of-function alleles provide new insight into understanding the multiple roles of Swi1-Swi3 in fork protection during DNA replication, and into understanding how replication forks are maintained in response to different genotoxic agents.

Novel relationships among DNA methylation, histone modifications and gene expression in Ascobolus.

By studying Ascobolus strains methylated in various portions of the native met2 gene or of the hph transgene, we generalized our previous observation that methylation of the downstream portion of a gene promotes its stable silencing and triggers the production of truncated transcripts which rarely extend through the methylated region. In contrast, methylation of the promoter region does not promote efficient gene silencing. The chromatin state of met2 methylated strains was investigated after partial micrococcal nuclease (MNase) digestion. We show that MNase sensitive sites present along the unmethylated regions are no longer observed along the methylated ones. These chromatin changes are not resulting from the absence of transcription. They are associated, in both met2 and hph, with modifications of core histones corresponding, on the N terminus of histone H3, to an increase of dimethylation of lysine 9 and a decrease of dimethylation of lysine 4. Contrary to other organisms, these changes are independent of the transcriptional state of the genes, and furthermore, no decrease in acetylation of histone H4 is observed in silenced genes.

Molecular and cellular dissection of mating-type switching steps in Schizosaccharomyces pombe.

A strand-specific imprint (break) controls mating-type switching in fission yeast. By introducing a thiamine repressible promoter upstream of the mat1 locus, we can force transcription through the imprinted region, erasing the imprint and inhibiting further mating-type switching, in a reversible manner. Starting from a synchronized, virgin M-cell population, we show that the site- and strand-specific break is formed when DNA replication intermediates appear at mat1 during the first S phase. The formation of the break is concomitant with a replication fork pause and binding of the Swi1 protein at mat1 until early G(2) and then rapidly disappears. Upon its formation, the break remains stable throughout the cell cycle and triggers mating-type switching during the second S phase. Finally, we have recreated the mating-type switching pedigree at the molecular and single-cell levels, allowing for the first time separation between the establishment of imprinting and its developmental fate.

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae.

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast.

Mating-type switching in the fission yeast Schizosaccharomyces pombe is initiated by a strand-specific imprint located at the mating-type (mat1) locus. We show that the imprint corresponds to a single-strand DNA break (SSB), which is site- but not sequence-specific. We identified three novel cis-acting elements, involved in the formation and stability of the SSB. One of these elements is essential for a replication fork pause next to mat1 and interacts in vivo with the Swi1 protein. Another element is essential for maintaining the SSB during cell cycle progression. These results suggest that the DNA break appears during the S-phase and is actively protected against repair. Consequently, during the following round of replication, a polar double-strand break is formed. We show that when the replication fork encounters the SSB, the leading-strand DNA polymerase is able to synthesize DNA to the edge of the SSB, creating a blunt-ended recombination intermediate.