RESUMO
BACKGROUND: Genome-wide association studies (GWAS) have revealed a multitude of candidate genetic variants affecting the risk of developing complex traits and diseases. However, the highlighted regions are typically in the non-coding genome, and uncovering the functional causative single nucleotide variants (SNVs) is challenging. Prioritization of variants is commonly based on genomic annotation with markers of active regulatory elements, but current approaches still poorly predict functional variants. To address this, we systematically analyze six markers of active regulatory elements for their ability to identify functional variants. RESULTS: We benchmark against molecular quantitative trait loci (molQTL) from assays of regulatory element activity that identify allelic effects on DNA-binding factor occupancy, reporter assay expression, and chromatin accessibility. We identify the combination of DNase footprints and divergent enhancer RNA (eRNA) as markers for functional variants. This signature provides high precision, but with a trade-off of low recall, thus substantially reducing candidate variant sets to prioritize variants for functional validation. We present this as a framework called FINDER-Functional SNV IdeNtification using DNase footprints and eRNA. CONCLUSIONS: We demonstrate the utility to prioritize variants using leukocyte count trait and analyze variants in linkage disequilibrium with a lead variant to predict a functional variant in asthma. Our findings have implications for prioritizing variants from GWAS, in development of predictive scoring algorithms, and for functionally informed fine mapping approaches.
Assuntos
Elementos Facilitadores Genéticos , RNAs Intensificadores , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Humanos , Pegada de DNA , Proteínas de Ligação a DNA/genética , Variação GenéticaRESUMO
How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Estrogênios , Regiões Promotoras Genéticas , Humanos , Cromatina/genética , Cromatina/metabolismo , Estrogênios/metabolismo , Transcrição Gênica , Células MCF-7 , Neoplasias da Mama/genética , Feminino , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Hibridização in Situ Fluorescente , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Ativação Transcricional , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismoRESUMO
The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.
Assuntos
Cromatina , Fatores de Transcrição , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Humanos , Fator de Transcrição CDX2/metabolismo , Fator de Transcrição CDX2/genética , Regulação da Expressão Gênica , Núcleo Celular/metabolismo , Sítios de Ligação , Montagem e Desmontagem da CromatinaRESUMO
Contacts between enhancers and promoters are thought to relate to their ability to activate transcription. Investigating factors that contribute to such chromatin interactions is therefore important for understanding gene regulation. Here, we have determined contact frequencies between millions of pairs of cis-regulatory elements from chromosome conformation capture data sets and analyzed a collection of hundreds of DNA-binding factors for binding at regions of enriched contacts. This analysis revealed enriched contacts at sites bound by many factors associated with active transcription. We show that active regulatory elements, independent of cohesin and polycomb, interact with each other across distances of tens of megabases in vertebrate and invertebrate genomes and that interactions correlate and change with activity. However, these ultra-long-range interactions are not dependent on RNA polymerase II transcription or individual transcription cofactors. Using simulations, we show that a model of chromatin and multivalent binding factors can give rise to long-range interactions via bridging-induced clustering. We propose that long-range interactions between cis-regulatory elements are driven by at least three distinct processes: cohesin-mediated loop extrusion, polycomb contacts, and clustering of active regions.
Assuntos
Cromatina , Sequências Reguladoras de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico/genética , Cromatina/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas do Grupo Polycomb/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Elementos Facilitadores Genéticos , Fator de Ligação a CCCTC/metabolismoRESUMO
Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci-at different length scales-can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.
Assuntos
Cromatina , Proteínas de Drosophila , Animais , Camundongos , Complexo Repressor Polycomb 1 , Núcleo Celular , Proteínas do Grupo PolycombRESUMO
Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size, and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional hubs, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harbouring distinct ecDNA species (EGFR, CDK4, PDGFRA). We find no evidence that ecDNA routinely cluster with one another or closely interact with transcriptional hubs. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy is similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that it is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with each other or with high concentrations of the transcriptional machinery.
Assuntos
Oncogenes , Células-Tronco , Humanos , Oncogenes/genética , DNARESUMO
With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.
Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Teste para COVID-19/economia , Humanos , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , SARS-CoV-2/genéticaRESUMO
The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.
Assuntos
Ciclo Celular/fisiologia , Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Divisão Celular/fisiologia , Linhagem Celular , Ácidos Indolacéticos/farmacologia , Interfase , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here, we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates in undirected differentiation. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.
Assuntos
Diferenciação Celular/genética , Fator 3 de Transcrição de Octâmero , Células-Tronco Pluripotentes/fisiologia , Fatores de Transcrição SOXB1 , Animais , Linhagem Celular , Endoderma/citologia , Endoderma/metabolismo , Elementos Facilitadores Genéticos/genética , Técnicas de Introdução de Genes/métodos , Camundongos , Placa Neural/citologia , Placa Neural/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
The transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-ß stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.
Assuntos
Regulação da Expressão Gênica , Expressão Gênica , Análise de Célula Única , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Transporte Biológico , Humanos , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measurements of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.
Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Interfase/fisiologia , Mitose/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Cromossomos/genética , DNA/genética , DNA/metabolismo , Humanos , Interfase/genética , Mitose/genética , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
The ability of some transcription factors to remain bound to specific genes on condensed mitotic chromosomes has been suggested to play a role in their rapid transcriptional reactivation upon mitotic exit. We have recently shown that SOX2 and OCT4 remain associated to mitotic chromosomes, and that depletion of SOX2 at the mitosis-G1 (M-G1) transition impairs its ability to maintain pluripotency and drive neuroectodermal commitment. Here we report on the role of SOX2 at the M-G1 transition in regulating transcriptional activity of embryonic stem cells. Using single cell time-lapse analysis of reporter constructs for STAT3 and SOX2/OCT4 activity, we show that SOX2/OCT4 do not lead to more rapid transcriptional reactivation in G1 than STAT3, a transcription factor that is excluded from mitotic chromosomes. We also report that only few endogenous target genes show decreased pre-mRNA levels after mitotic exit or in other cell cycle phases in the absence of SOX2 at the M-G1 transition. This suggests that bookmarked SOX2 target genes are not differently regulated than non-bookmarked target genes, and we discuss an alternative hypothesis on how mitotic bookmarking by SOX2 and other sequence-specific transcription factors could be involved in transcriptional regulation.
Assuntos
Regulação da Expressão Gênica , Mitose/genética , Fatores de Transcrição SOXB1/metabolismo , Transcrição Gênica , Animais , Éxons/genética , Íntrons/genética , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Nocodazol/farmacologia , Plasmídeos/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.
Assuntos
Diferenciação Celular/genética , Mitose/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Reprogramação Celular/genética , Cromatina/metabolismo , Células-Tronco Embrionárias , Fase G1 , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células NIH 3T3 , Placa Neural/citologia , Placa Neural/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação ProteicaRESUMO
By employing a genetic selection that forces the cell to fold an unstable, aggregation-prone test protein in order to survive, we have generated bacterial strains with enhanced periplasmic folding capacity. These strains enhance the soluble steady-state level of the test protein. Most of the bacterial variants we isolated were found to overexpress one or more periplasmic proteins including OsmY, Ivy, DppA, OppA, and HdeB. Of these proteins, only HdeB has convincingly been previously shown to function as chaperone in vivo. By giving bacteria the stark choice between death and stabilizing a poorly folded protein, we have now generated designer bacteria selected for their ability to stabilize specific proteins.