Pancreatic acinar cells rely on PTF1 and other transcription factors to deploy their transcriptional program. We identify NFIC as a NR5A2 interactor and regulator of acinar differentiation. NFIC ...binding sites are enriched in NR5A2 ChIP-Sequencing peaks. Nfic knockout mice have a smaller, histologically normal, pancreas with reduced acinar gene expression. NFIC binds and regulates the promoters of acinar genes and those involved in RNA/protein metabolism, and Nfic knockout pancreata show defective ribosomal RNA maturation. NFIC dampens the endoplasmic reticulum stress program through binding to gene promoters and is required for resolution of Tunicamycin-mediated stress. NFIC is down-regulated during caerulein pancreatitis and is required for recovery after damage. Normal human pancreata with low levels of NFIC transcripts display reduced expression of genes down-regulated in Nfic knockout mice. NFIC expression is down-regulated in mouse and human pancreatic ductal adenocarcinoma. Consistently, Nfic knockout mice develop a higher number of mutant Kras-driven pre-neoplastic lesions.
The Wnt signaling pathway is an important cellular mechanism for regulating differentiation processes as well as cell cycle events, and different inhibitors of this pathway, for example, PRI-724, are ...showing promising results in clinical trials for treatment of advanced pancreatic adenocarcinoma or ovarian cancer. Growing evidence suggests that Wnt signaling may also be crucial for tumorigenesis and progression of soft tissue sarcomas (STS), a malignant neoplasm with few therapeutic options at an advanced state. Our study with several STS cell lines and primary cultures shows that inhibition of Wnt/β-catenin signaling with PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates. TCF/β-catenin-mediated transcriptional activity is decreased in treated cells, leading to downregulation of its target genes CCND1 and CDC25A. The latter was critical because its downregulation via siRNA was able to mimic the effect of PRI-724 on cell cycle arrest and cell death induction. An evaluation of NCBI/GenBank data confirmed that CDC25A mRNA is elevated in STS patients. Importantly, PRI-724 in combination with standard STS chemotherapeutics doxorubicin or trabectedin enhanced their antitumoral effect in a synergistic manner according to isobolographic analysis, suggesting that Wnt inhibition through PRI-724 could be a beneficial combination regime in patients with advanced STS.
Genes can encode multiple isoforms, broadening their functions and providing a molecular substrate to evolve phenotypic diversity. Evolution of isoform function is a potential route to adapt to new ...environments. Here we show that de novo, beneficial alleles in the
gene became fixed in two laboratory lineages of
after isolation from the wild in 1951, before methods of cryopreservation were developed.
encodes an ortholog of BPTF, a large (>300 kD) multidomain subunit of the NURF chromatin remodeling complex. Using CRISPR-Cas9 genome editing and transgenic rescue, we demonstrate that in
,
has split into two, largely non-overlapping isoforms (NURF-1.D and NURF-1.B, which we call Yin and Yang, respectively) that share only two of 26 exons. Both isoforms are essential for normal gametogenesis but have opposite effects on male/female gamete differentiation. Reproduction in hermaphrodites, which involves production of both sperm and oocytes, requires a balance of these opposing Yin and Yang isoforms. Transgenic rescue and genetic position of the fixed mutations suggest that different isoforms are modified in each laboratory strain. In a related clade of
nematodes, the shared exons have duplicated, resulting in the split of the Yin and Yang isoforms into separate genes, each containing approximately 200 amino acids of duplicated sequence that has undergone accelerated protein evolution following the duplication. Associated with this duplication event is the loss of two additional
transcripts, including the long-form transcript and a newly identified, highly expressed transcript encoded by the duplicated exons. We propose these lost transcripts are non-functional side products necessary to transcribe the Yin and Yang transcripts in the same cells. Our work demonstrates how gene sharing, through the production of multiple isoforms, can precede the creation of new, independent genes.
Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression. Dealing with high levels of transcription during replication is critical to prevent ...collisions between the transcription and replication pathways and avoid recombination events. In response to osmostress, hundreds of stress-responsive genes are rapidly induced by the stress-activated protein kinase (SAPK) Hog1 (ref. 6), even during S phase. Here we show in Saccharomyces cerevisae that a single signalling molecule, Hog1, coordinates both replication and transcription upon osmostress. Hog1 interacts with and phosphorylates Mrc1, a component of the replication complex. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage. Mrc1 phosphorylation by Hog1 delays early and late origin firing by preventing Cdc45 loading, as well as slowing down replication-complex progression. Regulation of Mrc1 by Hog1 is completely independent of Mec1 and Rad53. Cells carrying a non-phosphorylatable allele of MRC1 (mrc1(3A)) do not delay replication upon stress and show a marked increase in transcription-associated recombination, genomic instability and Rad52 foci. In contrast, mrc1(3A) induces Rad53 and survival in the presence of hydroxyurea or methyl methanesulphonate. Therefore, Hog1 and Mrc1 define a novel S-phase checkpoint independent of the DNA-damage checkpoint that permits eukaryotic cells to prevent conflicts between DNA replication and transcription, which would otherwise lead to genomic instability when both phenomena are temporally coincident.
Transcription is a major contributor to genome instability. A main cause of transcription‐associated instability relies on the capacity of transcription to stall replication. However, we know little ...of the possible role, if any, of the RNA polymerase (RNAP) in this process. Here, we analyzed 4 specific yeast RNAPII mutants that show different phenotypes of genetic instability including hyper‐recombination, DNA damage sensitivity and/or a strong dependency on double‐strand break repair functions for viability. Three specific alleles of the RNAPII core, rpb1‐1, rpb1‐S751F and rpb9∆, cause a defect in replication fork progression, compensated for by additional origin firing, as the main action responsible for instability. The transcription elongation defects of rpb1‐S751F and rpb9∆ plus our observation that rpb1‐1 causes RNAPII retention on chromatin suggest that RNAPII could participate in facilitating fork progression upon a transcription‐replication encounter. Our results imply that the RNAPII or ancillary factors actively help prevent transcription‐associated genome instability.
Synopsis
Collisions between RNA transcription and DNA replication machineries impair replication fork (RF) progression and cause DNA breaks, demanding homologous recombination for repair and RF restart. Identification and characterization of specific yeast RNA polymerase II mutations that enhance DNA RF stalling and trigger hyper‐recombination suggests that increased RNAP II retention at transcription sites may contribute to genomic instability and that eukaryotic RNAP II is more than a passive entity in the management of transcription–replication encounters.
Specific RNAP II mutants in Saccharomyces cerevisiae, rpb1‐1, rpb1S71F and rbp9Δ show an increase in genetic instability as detected by hyper‐recombination, DNA damage sensitivity, and dependency on DSB repair functions for viability.
Impaired RF progression in these RNAP II mutants can be compensated by additional origin firing.
Under replication stress, checkpoint‐activating DNA lesions accumulate in these mutant cells and are repaired only with considerable delay.
RNase H1 overexpression does not suppress genome instability in rpb1 mutants, indicating that an increase in R‐loops is not a main determinant of RF stalling.
The causative rpb1‐1 mutation increases retention of RNAP II at the site of transcription.
Identification of specific yeast RNA polymerase II mutations that cause genomic instability by enhancing collisions with the DNA replication machinery suggests that eukaryotic RNAP II is more than a passive entity in the management of transcription–replication encounters.
MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Selected sarcoma subtypes are being treated with Trabectedin in second line, which promotes DNA damage and p53-dependent ...apoptosis. The aim of this study was to evaluate the improvement of Trabectedin response with MDM2 inhibitors in soft tissue sarcomas. The antitumor effects of Trabectedin, Nutlin-3A and RG7112 as single agents or in combination were examined in vitro. RG7112 significantly synergized with Trabectedin in MDM2-amplified liposarcoma cells, representing a promising new therapeutic strategy for the treatment of sarcomas with MDM2 amplification.
Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin and represent around 1% of adult cancers, being a very heterogeneous group of tumors with more than 50 different subtypes. The ...Wnt signaling pathway is involved in the development and in the regulation, self-renewal, and differentiation of mesenchymal stem cells, and plays a role in sarcomagenesis. In this study, we have tested pharmacologic inhibition of Wnt signaling mediated by disruption of TCF/β-catenin binding and AXIN stabilization, being the first strategy more efficient in reducing cell viability and downstream effects. We have shown that disruption of TCF/β-catenin binding with PKF118-310 produces
antitumor activity in a panel of prevalent representative STS cell lines and primary cultures. At the molecular level, PKF118-310 treatment reduced β-catenin nuclear localization, reporter activity, and target genes, resulting in an increase in apoptosis. Importantly, combination of PKF118-310 with doxorubicin resulted in enhanced reduction of cell viability, suggesting that Wnt inhibition could be a new combination regime in these patients. Our findings support the usefulness of Wnt inhibitors as new therapeutic strategies for the prevalent STS.
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Chronic inflammation increases the risk of developing one of several types of cancer. Inflammatory responses are currently thought to be controlled by mechanisms that rely on transcriptional networks ...that are distinct from those involved in cell differentiation. The orphan nuclear receptor NR5A2 participates in a wide variety of processes, including cholesterol and glucose metabolism in the liver, resolution of endoplasmic reticulum stress, intestinal glucocorticoid production, pancreatic development and acinar differentiation. In genome-wide association studies, single nucleotide polymorphisms in the vicinity of NR5A2 have previously been associated with the risk of pancreatic adenocarcinoma. In mice, Nr5a2 heterozygosity sensitizes the pancreas to damage, impairs regeneration and cooperates with mutant Kras in tumour progression. Here, using a global transcriptomic analysis, we describe an epithelial-cell-autonomous basal pre-inflammatory state in the pancreas of Nr5a2
mice that is reminiscent of the early stages of pancreatitis-induced inflammation and is conserved in histologically normal human pancreases with reduced expression of NR5A2 mRNA. In Nr5a2
mice, NR5A2 undergoes a marked transcriptional switch, relocating from differentiation-specific to inflammatory genes and thereby promoting gene transcription that is dependent on the AP-1 transcription factor. Pancreatic deletion of Jun rescues the pre-inflammatory phenotype, as well as binding of NR5A2 to inflammatory gene promoters and the defective regenerative response to damage. These findings support the notion that, in the pancreas, the transcriptional networks involved in differentiation-specific functions also suppress inflammatory programmes. Under conditions of genetic or environmental constraint, these networks can be subverted to foster inflammation.
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