The transcription factor SRF (serum response factor) recruits two families of coactivators, the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors), to couple gene ...transcription to growth factor signaling. Here we investigated the role of the SRF network in the immediate transcriptional response of fibroblasts to serum stimulation. SRF recruited its cofactors in a gene-specific manner, and virtually all MRTF binding was directed by SRF. Much of SRF DNA binding was serum-inducible, reflecting a requirement for MRTF-SRF complex formation in nucleosome displacement. We identified 960 serum-responsive SRF target genes, which were mostly MRTF-controlled, as assessed by MRTF chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) and/or sensitivity to MRTF-linked signals. MRTF activation facilitates RNA polymerase II (Pol II) recruitment or promoter escape according to gene context. MRTF targets encode regulators of the cytoskeleton, transcription, and cell growth, underpinning the role of SRF in cytoskeletal dynamics and mechanosensing. Finally, we show that specific activation of either MRTFs or TCFs can reset the circadian clock.
The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in ...the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression.
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•Integrated ChIP-seq Hi-C analysis identifies over 700 TCF-dependent SRF target genes•Over 60% of TPA-inducible gene transcription is TCF-dependent•TCF-dependent transcription potentiates cell proliferation•TCF/MRTF competition for SRF determines contractility and pro-invasive behavior
Two cofactor families, the TCFs and the MRTFs, compete for binding to SRF, a key regulator of the mitogen-responsive transcription. TCFs not only control proliferative gene expression programs but also regulate access of the MRTFs to SRF, thereby regulating cell contractility and pro-invasive behavior.
The cohesin complex is at the heart of many chromosomal activities, including sister chromatid cohesion and transcriptional regulation. Cohesin loading onto chromosomes depends on the Scc2-Scc4 ...cohesin loader complex, but the chromatin features that form cohesin loading sites remain poorly understood. Here we show that the RSC chromatin remodeling complex recruits budding yeast Scc2-Scc4 to broad nucleosome-free regions, which the cohesin loader helps to maintain. Consequently, inactivation of either the cohesin loader or the RSC complex has similar effects on nucleosome positioning, gene expression and sister chromatid cohesion. These results show an intimate link between local chromatin structure and higher-order chromosome architecture. Our findings pertain to the similarities between two severe human disorders, Cornelia de Lange syndrome, which is caused by alterations in the human cohesin loader, and Coffin-Siris syndrome, which results from alterations in human RSC complex components. Both syndromes can arise from gene misregulation due to related changes in the nucleosome landscape.
Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ...ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73-75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development.
The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to activate the innate immune system. Here, we ...report the unexpected discovery that cGAS also senses dysfunctional protein production. Purified ribosomes interact directly with cGAS and stimulate its DNA-dependent activity in vitro. Disruption of the ribosome-associated protein quality control (RQC) pathway, which detects and resolves ribosome collision during translation, results in cGAS-dependent ISG expression and causes re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and orthogonal perturbations that result in elevated levels of collided ribosomes and RQC activation cause sub-cellular re-localization of cGAS and ribosome binding in vivo as well. Thus, translation stress potently increases DNA-dependent cGAS activation. These findings have implications for the inflammatory response to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.
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•RQC factors involved in disassembling collided ribosomes suppress the cGAS pathway•Ribosomes interact with cGAS, stimulating its DNA-dependent activity•cGAS preferentially interacts with collided ribosomes•Ribosome collision leads to re-localization of cGAS to the cytosol and ISG activation
Wan et al. show that cGAS, a well-known DNA sensor, can also sense translation stress by direct interaction with ribosomes, which in turn induces accumulation of cGAS in the cytosol, stimulation of its DNA-dependent catalytic activity, and activation of innate immunity signaling via ISG activation.
Genetic analysis was applied to different regions of renal-cell cancers. The lesions noted in the tumor were not found in every sample, and regions of the tumor had different gene-expression ...patterns. This suggests that extrapolation from results of a single biopsy may be problematic.
Large-scale sequencing analyses of solid cancers have identified extensive heterogeneity between individual tumors.
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Genetic intratumor heterogeneity has also been shown
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and can contribute to treatment failure and drug resistance. Intratumor heterogeneity may have important consequences for personalized-medicine approaches that commonly rely on single tumor-biopsy samples to portray tumor mutational landscapes. Studies comparing mutational profiles of primary tumors and associated metastatic lesions
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or local recurrences
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have provided evidence of intratumor heterogeneity at nucleotide resolution. Intratumor heterogeneity within primary tumors and associated metastatic sites has not been systematically characterized by next-generation sequencing. We applied exome sequencing, chromosome aberration analysis, . . .
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide ...transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery—persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
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•Specific RPB1 K1268 ubiquitylation targets RNAPII for UV-induced proteolysis•RPB1 K1268 ubiquitylation is required for surviving DNA damage•Control of the RNAPII pool via degradation regulates the transcriptome after UV•Lack of transcription recovery in Cockayne syndrome is caused by unstable RNAPII
Control of the pool of available RNA polymerase II shapes how cells respond to UV stress and the efficacy of the resulting damage response.
Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative ...polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.
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•Human SCAF4 and SCAF8 couple RNAPII Ser2P- and Ser5P-binding and RNA processing•SCAF4 and SCAF8 bind nascent RNA upstream of early polyadenylation sites•SCAF4 and SCAF8 prevent early mRNA transcript cleavage and polyadenylation•Lack of SCAF4 and SCAF8 result in truncated protein products and is cell lethal
Eukaryotic anti-terminator proteins suppress usage of early polyadenylation sites to prevent production of truncated proteins.
Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental ...settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1.
Chromosomal instability (CIN) contributes to cancer evolution, intratumor heterogeneity, and drug resistance. CIN is driven by chromosome segregation errors and a tolerance phenotype that permits the ...propagation of aneuploid genomes. Through genomic analysis of colorectal cancers and cell lines, we find frequent loss of heterozygosity and mutations in BCL9L in aneuploid tumors. BCL9L deficiency promoted tolerance of chromosome missegregation events, propagation of aneuploidy, and genetic heterogeneity in xenograft models likely through modulation of Wnt signaling. We find that BCL9L dysfunction contributes to aneuploidy tolerance in both TP53-WT and mutant cells by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID. Efforts to exploit aneuploidy tolerance mechanisms and the BCL9L/caspase-2/BID axis may limit cancer diversity and evolution.
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•Loss-of-function alterations in BCL9L are frequent in aneuploid CRC•BCL9L dysfunction drives aneuploidy tolerance by reducing levels of caspase-2•Caspase-2 activation following aneuploidy results in MDM2 and BID cleavage•p53 stabilization after chromosome missegregation is regulated by caspase-2
López-García et al. find that BCL9L is often genetically inactivated in human colorectal cancers with chromosomal instability. BCL9L dysfunction promotes aneuploidy tolerance by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID independent of TP53 mutation status.
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