Genome-wide surveillance of transcription errors in response to genotoxic stress.

Mutagenic compounds are a potent source of human disease. By inducing genetic instability, they can accelerate the evolution of human cancers or lead to the development of genetically inherited diseases. Here, we show that in addition to genetic mutations, mutagens are also a powerful source of transcription errors. These errors arise in dividing and nondividing cells alike, affect every class of transcripts inside cells, and, in certain cases, greatly exceed the number of mutations that arise in the genome. In addition, we reveal the kinetics of transcription errors in response to mutagen exposure and find that DNA repair is required to mitigate transcriptional mutagenesis after exposure. Together, these observations have far-reaching consequences for our understanding of mutagenesis in human aging and disease, and suggest that the impact of DNA damage on human physiology has been greatly underestimated.

Functional evidence implicating FOXL2 in non-syndromic premature ovarian failure and in the regulation of the transcription factor OSR2.

BACKGROUND: FOXL2 encodes a forkhead transcription factor whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), involving craniofacial/palpebral abnormalities often associated with premature ovarian failure (POF). RESULTS: We describe a FOXL2 variant (p.Gly187Asp) in a case of POF without BPES. The subcellular localisation of FOXL2-G187D was normal but its transactivation capacity tested on two reporter promoters, one of which should be relevant to the ovary, was significantly lower than that of normal FOXL2. However, FOXL2-G187D was able to activate strongly a reporter construct driven by the promoter of Osr2 (odd-skipped related 2 transcription factor), which we have suggested to be a crucial target of FOXL2 in the craniofacial region. This is compatible with the absence of BPES in our patient. CONCLUSIONS: Our data provide evidence in favour of the implication of FOXL2 variants in non-syndromic POF and confirm the regulatory interaction between FOXL2 and OSR2 whose perturbation might contribute to the palpebral abnormalities observed in BPES patients.

The mutations and potential targets of the forkhead transcription factor FOXL2.

Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). This genetic disorder is characterized by eyelid and mild craniofacial abnormalities that can appear associated with premature ovarian failure. FOXL2 is one of the earliest ovarian markers and it offers, along with its targets, an excellent model to study ovarian development and function in normal and pathological conditions. In this review we summarize recent data concerning FOXL2, its mutations and its potential targets. Indeed, many mutations have been described in the coding sequence of FOXL2. Among them, polyalanine expansions and premature nonsense mutations have been shown to induce protein aggregation. In the context of the ovary, FOXL2 has been suggested to be involved in the regulation of cholesterol and steroid metabolism, apoptosis, reactive oxygen species detoxification and inflammation processes. The elucidation of the impact of FOXL2 mutations on its function will allow a better understanding of the pathogenic mechanisms underlying the BPES phenotype.

Mos activates myogenic differentiation by promoting heterodimerization of MyoD and E12 proteins.

The activities of myogenic basic helix-loop-helix (bHLH) factors are regulated by a number of different positive and negative signals. Extensive information has been published about the molecular mechanisms that interfere with the process of myogenic differentiation, but little is known about the positive signals. We previously showed that overexpression of rat Mos in C2C12 myoblasts increased the expression of myogenic markers whereas repression of Mos products by antisense RNAs inhibited myogenic differentiation. In the present work, our results show that the rat mos proto-oncogene activates transcriptional activity of MyoD protein. In transient transfection assays, Mos promotes transcriptional transactivation by MyoD of the muscle creatine kinase enhancer and/or a reporter gene linked to MyoD-DNA binding sites. Physical interaction between Mos and MyoD, but not with E12, is demonstrated in vivo by using the two-hybrid approach with C3H10T1/2 cells and in vitro by using the glutathione S-transferase (GST) pull-down assays. Unphosphorylated MyoD from myogenic cell lysates and/or bacterially expressed MyoD physically interacts with Mos. This interaction occurs via the helix 2 region of MyoD and a highly conserved region in Mos proteins with 40% similarity to the helix 2 domain of the E-protein class of bHLH factors. Phosphorylation of MyoD by activated GST-Mos protein inhibits the DNA-binding activity of MyoD homodimers and promotes MyoD-E12 heterodimer formation. These data support a novel function for Mos as a mediator (coregulator) of muscle-specific gene(s) expression.

Overexpression of Mos(rat) proto-oncogene product enhances the positive autoregulatory loop of MyoD.

The myogenic b-HLH transcription factor MyoD activates expression of muscle-specific genes and autoregulates positively its own expression. Various factors such as growth factors and oncogene products repress transcriptional activity of MyoD. The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate myogenic differentiation by specific phosphorylation of MyoD which favors heterodimerization of MyoD and E12 proteins. Here we show that overexpression of Mos enhances the expression level of MyoD protein in myoblasts although phosphorylation of MyoD by Mos does not modify its stability but promotes transcriptional transactivation of the MyoD promoter linked to the luciferase reporter gene. Moreover, co-expression of MyoD with Mos(wt) but not with the kinase-inactive Mos(KM) greatly enhances expression of endogenous MyoD protein and the DNA binding activity of MyoD/E12 heterodimers in 10T1/2 cells. Our data suggest that Mos increases the ability of MyoD to transactivate both muscle-specific genes and its own promoter and could therefore participate in the positive autoregulation loop of MyoD and muscle differentiation.

Adult ovarian granulosa cell tumor transcriptomics: prevalence of FOXL2 target genes misregulation gives insights into the pathogenic mechanism of the p.Cys134Trp somatic mutation.

Ovarian granulosa cell tumors (OGCT) are the most frequent kind of sex cord-stromal tumors, and represent ∼2-5% of all ovarian malignancies. OGCTs exist as two entities, juvenile and adult types, with specific clinical and pathological characteristics. The molecular pathogenesis of these tumors has just begun to be unraveled. Indeed, recent studies have indicated that mutation and/or misregulation of the key ovarian transcription factor FOXL2 has a role in OGCT formation, although the mechanisms remain unclear. To better understand the molecular characteristics of OGCT, we studied the transcriptomic profiles of ten human adult-type OGCT samples, as well as ethnically matched granulosa cell (GC) controls. We find that the OGCT samples analyzed herein exhibit several hallmarks of cancer, including increased expression of genes linked to cell proliferation, but decreased expression of those conferring sensitivity to cell death. Moreover, genes differentially expressed in OGCTs are significantly enriched for known FOXL2 target genes, consistently with the prevalence of FOXL2 somatic mutation in these tumors. Expression of these targets is altered in a way expected to promote malignant transformation, for instance, through induction of genes associated with faster cell cycling and downregulation of genes associated with cell death. Over time, such defects may be responsible at least partly for the malignant transformation of healthy GCs into OGCT. These insights into the molecular pathogenesis of OGCTs may open the way to new efforts in the development of more targeted therapeutic strategies for OGCT patients.

Rat seminal vesicle FAD-dependent sulfhydryl oxidase. Biochemical characterization and molecular cloning of a member of the new sulfhydryl oxidase/quiescin Q6 gene family.

Rat FAD-dependent sulfhydryl oxidase was purified; partial sequencing indicated that it was homologous to human quiescin Q6. A cDNA (GenBank accession no. AF285078) was cloned from rat seminal vesicles, and active recombinant sulfhydryl oxidase was expressed in Chinese hamster ovary epithelial cells. This 2472-nucleotide cDNA has an open reading frame of 1710 base pairs, encoding a protein of 570 amino acids including a 32-amino acid leader sequence and two potential sites for N-glycosylation. One of them is used and the 64,000 M(r) purified protein was transformed to 61,000 by the action of endoglycosidase F. Northern blotting and reverse transcription-polymerase chain reaction analyses showed that there were small amounts of sulfhydryl oxidase in the rat testis, prostate, lung, heart, kidney, spleen, and liver, and that the gene was highly expressed in seminal vesicles and epididymis. Rat sulfhydryl oxidase cDNA corresponds to the human cell growth inhibiting factor cDNA, which could be a differently spliced form of quiescin Q6. Comparing sulfhydryl oxidase sequences with those of human quiescin Q6 and mammalian and Caenorhabditis elegans quiescin Q6-related genes established the existence of a new family of FAD-dependent sulfhydryl oxidase/quiescin Q6-related genes containing protein-disulfide isomerase-type thioredoxin and yeast ERV1 domains.