RESUMEN
In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.
Asunto(s)
Actinas/metabolismo , Ritmo Circadiano , Regulación de la Expresión Génica , Técnicas Genéticas , Factor de Respuesta Sérica/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Humanos , Masculino , Ratones , Proteínas Circadianas Period/metabolismo , Ratas , Factores de Transcripción/metabolismoRESUMEN
The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3' direction several hundred kilobases downstream of genes. The YFFF mutant shows reduced Pol II at promoter-proximal pause sites, a loss of interaction with the Mediator and Integrator complexes, and impaired recruitment of these complexes to chromatin. Consistent with these observations, Pol II loading at enhancers and maturation of snRNAs are altered in the YFFF context genome-wide. We conclude that tyrosine1 residues of the CTD control termination of transcription by Pol II.
Asunto(s)
ARN Polimerasa II/genética , ARN Mensajero/biosíntesis , Terminación de la Transcripción Genética/fisiología , Transcripción Genética/fisiología , Tirosina/genética , Línea Celular Tumoral , Cromatina/metabolismo , Humanos , Mutación/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genéticaRESUMEN
The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.
Asunto(s)
Proteínas de Unión al Calcio , Diferenciación Celular , Impresión Genómica , ARN Largo no Codificante , Animales , Ratones , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Metilación de ADN , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Proteínas Nucleares , Proteínas Gestacionales , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
The largest subunit of RNA polymerase (Pol) II harbors an evolutionarily conserved C-terminal domain (CTD), composed of heptapeptide repeats, central to the transcriptional process. Here, we analyze the transcriptional phenotypes of a CTD-Δ5 mutant that carries a large CTD truncation in human cells. Our data show that this mutant can transcribe genes in living cells but displays a pervasive phenotype with impaired termination, similar to but more severe than previously characterized mutations of CTD tyrosine residues. The CTD-Δ5 mutant does not interact with the Mediator and Integrator complexes involved in the activation of transcription and processing of RNAs. Examination of long-distance interactions and CTCF-binding patterns in CTD-Δ5 mutant cells reveals no changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription in living cells. We propose a model in which CTD-depleted Pol II has a lower entry rate onto DNA but becomes pervasive once engaged in transcription, resulting in a defect in termination.
Asunto(s)
ARN Polimerasa II , Transcripción Genética , Humanos , ARN Polimerasa II/metabolismo , Núcleo Celular/metabolismo , Mutación , FosforilaciónRESUMEN
We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Histonas/metabolismo , Factor de Respuesta Sérica/metabolismo , Factores de Transcripción TCF/metabolismo , Transcripción Genética , Animales , Línea Celular , Cromatina/efectos de los fármacos , Cromatina/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Activación Enzimática , Ratones , Ratones Noqueados , Mutación , Fosforilación , Interferencia de ARN , Factor de Respuesta Sérica/genética , Transducción de Señal , Factores de Transcripción TCF/genética , Acetato de Tetradecanoilforbol/farmacología , Sitio de Iniciación de la Transcripción , Transcripción Genética/efectos de los fármacos , Transfección , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
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.
Asunto(s)
Fibroblastos/metabolismo , Regulación de la Expresión Génica , Factor de Respuesta Sérica/genética , Factores Complejos Ternarios/genética , Animales , Secuencia de Bases , Línea Celular , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Humanos , Ratones , Factor de Respuesta Sérica/metabolismo , Transducción de Señal , Factores Complejos Ternarios/antagonistas & inhibidores , Factores Complejos Ternarios/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 ß and γ isoforms (HP1ß/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K-AKT-mTOR axis and switch to glycolysis. While differentiation of naive TRIM28-/- T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28-/- regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1ß/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.
Asunto(s)
Diferenciación Celular/fisiología , Reprogramación Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética/fisiología , Linfocitos T/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Animales , Autoinmunidad/fisiología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Plasticidad de la Célula/fisiología , Reprogramación Celular/genética , Homólogo de la Proteína Chromobox 5 , Colon/patología , Citocinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Silenciador del Gen , Histonas/metabolismo , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transcriptoma , Proteína 28 que Contiene Motivos Tripartito/genéticaRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Fibroblastos/fisiología , Suero/metabolismo , Transducción de Señal , Transcripción Genética/genética , Animales , Proteínas CLOCK/genética , Relojes Circadianos/genética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Factor de Respuesta Sérica/metabolismoRESUMEN
Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF-dependent transcriptional program that supports survival of epithelial cells.
Asunto(s)
Apoptosis/fisiología , Células Epiteliales/metabolismo , Glándulas Mamarias Humanas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Respuesta Sérica/metabolismo , Transcripción Genética/fisiología , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Células Epiteliales/citología , Femenino , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Glándulas Mamarias Humanas/citología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Factor de Respuesta Sérica/genéticaRESUMEN
AIMS: To investigate the effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors vs. dipeptidyl peptidase-4 (DPP-4) inhibitors on renal function preservation (RFP) using real-world data of patients with type 2 diabetes in Japan, and to identify which subgroups of patients obtained greater RFP benefits with SGLT2 inhibitors vs. DPP-4 inhibitors. METHODS: We retrospectively analysed claims data recorded in the Medical Data Vision database in Japan of patients with type 2 diabetes (aged ≥18 years) prescribed any SGLT2 inhibitor or any DPP-4 inhibitor between May 2014 and September 2016 (identification period), in whom estimated glomerular filtration rate (eGFR) was measured at least twice (baseline, up to 6 months before the index date; follow-up, 9 to 15 months after the index date) with continuous treatment until the follow-up eGFR. The endpoint was the percentage of patients with RFP, defined as no change or an increase in eGFR from baseline to follow-up. A proprietary supervised learning algorithm (Q-Finder; Quinten, Paris, France) was used to identify the profiles of patients with an additional RFP benefit of SGLT2 inhibitors vs. DPP-4 inhibitors. RESULTS: Data were available for 990 patients prescribed SGLT2 inhibitors and 4257 prescribed DPP-4 inhibitors. The proportion of patients with RFP was significantly greater in the SGLT2 inhibitor group (odds ratio 1.27; P = 0.01). The Q-Finder algorithm identified four clinically relevant subgroups showing superior RFP with SGLT2 inhibitors (P < 0.1): no hyperlipidaemia and eGFR ≥79 mL/min/1.73 m2 ; eGFR ≥79 mL/min/1.73 m2 and diabetes duration ≤1.2 years; eGFR ≥75 mL/min/1.73 m2 and use of antithrombotic agents; and haemoglobin ≤13.4 g/dL and LDL cholesterol ≥95.1 mg/dL. In each profile, glycaemic control was similar in the two groups. CONCLUSION: SGLT2 inhibitors were associated with more favourable RFP vs. DPP-4 inhibitors in patients with certain profiles in real-world settings in Japan.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Hipoglucemiantes/uso terapéutico , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Adulto , Algoritmos , Bases de Datos Factuales , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Humanos , Japón , Riñón/efectos de los fármacos , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Aprendizaje Automático Supervisado , Resultado del TratamientoRESUMEN
Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells (HSCs) during development. Serum response factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the myocardin-related transcription factor (MRTF) family of G-actin-regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact but does not become established in fetal bone marrow. Srf-null HSC progenitor cells (HSC/Ps) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarize in response to SDF-1 and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. Srf-null HSC/Ps exhibit substantial deficits in cytoskeletal gene expression. MRTF-SRF signaling is thus critical for expression of genes required for the response to chemokine signaling during hematopoietic development.
Asunto(s)
Médula Ósea/embriología , Médula Ósea/fisiología , Células Madre Hematopoyéticas/fisiología , Factor de Respuesta Sérica/fisiología , Nicho de Células Madre , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Médula Ósea/crecimiento & desarrollo , Movimiento Celular/genética , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genética , Nicho de Células Madre/genéticaRESUMEN
In vitro, without Mediator, the association of general transcription factors (GTF) and RNA polymerase II (Pol II) in preinitiation complexes (PIC) occurs in an orderly fashion. In this work, we explore the in vivo function of Mediator in GTF recruitment to PIC. A direct interaction between Med11 Mediator head subunit and Rad3 TFIIH subunit was identified. We explored the significance of this interaction and those of Med11 with head module subunits Med17 and Med22 and found that impairing these interactions could differentially affect the recruitment of TFIIH, TFIIE, and Pol II in the PIC. A med11 mutation that altered promoter occupancy by the TFIIK kinase module of TFIIH genome-wide also reduced Pol II CTD serine 5 phosphorylation. We conclude that the Mediator head module plays a critical role in TFIIH and TFIIE recruitment to the PIC. We identify steps in PIC formation that suggest a branched assembly pathway.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , ADN Helicasas/metabolismo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico/genética , Complejo Mediador , Modelos Biológicos , Mutación/genética , Fosforilación , Fosfotransferasas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción TFII/metabolismoRESUMEN
To combat cancer, a comprehensive understanding of the molecular mechanisms and behaviors involved in carcinogenesis is crucial, as tumorigenesis is a complex process influenced by various genetic events and disease hallmarks. The B-MYB gene encodes a transcription factor involved in cell cycle regulation, survival, and differentiation in normal cells. B-MYB can be transformed into an oncogene through mutations, and abnormal expression of B-MYB has been identified in various cancers, including lung cancer, and is associated with poor prognosis. Targeting this oncogene is a promising approach for anti-cancer drug design. B-MYB has been deemed undruggable in previous reports, necessitating the search for novel therapeutic options. In this study, we found that the B-MYB gene promoter contains several G/C rich motifs compatible with G-quadruplex (G4) formation. We investigated and validated the existence of G4 structures in the promoter region of B-MYB, first in vitro using a combination of bioinformatics, biophysical, and biochemical methods, then in cell with the recently developed G4access method.
Asunto(s)
G-Cuádruplex , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Regiones Promotoras Genéticas/genética , Humanos , Transactivadores/genética , Transactivadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Motivos de Nucleótidos/genéticaRESUMEN
ABSTRACT: Real-world data (RWD) are essential to complement clinical trial (CT) data, but major challenges remain, such as data quality. REal world dAta in LYmphoma and Survival in Adults (REALYSA) is a prospective noninterventional multicentric cohort started in 2018 that included patients newly diagnosed with lymphoma in France. Herein is a proof-of-concept analysis on patients with first-line diffuse large B-cell lymphoma (DLBCL) to (1) evaluate the capacity of the cohort to provide robust data through a multistep validation process; (2) assess the consistency of the results; and (3) conduct an exploratory transportability assessment of 2 recent phase 3 CTs (POLARIX and SENIOR). The analysis population comprised 645 patients with DLBCL included before 31 March 2021 who received immunochemotherapy and for whom 3589 queries were generated, resulting in high data completeness (<4% missing data). Median age was 66 years, with mostly advanced-stage disease and high international prognostic index (IPI) score. Treatments were mostly rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine, and prednisone (R-CHOP 75%) and reduced dose R-CHOP (13%). Estimated 1-year event-free survival (EFS) and overall survival rates were 77.9% and 90.0%, respectively (median follow-up, 9.9 months). Regarding transportability, when applying the CT's main inclusion criteria (age, performance status, and IPI), outcomes seemed comparable between patients in REALYSA and standard arms of POLARIX (1-year progression-free survival 79.8% vs 79.8%) and SENIOR (1-year EFS, 64.5% vs 60.0%). With its rigorous data validation process, REALYSA provides high-quality RWD, thus constituting a platform for numerous scientific purposes. The REALYSA study was registered at www.clinicaltrials.gov as #NCT03869619.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma de Células B Grandes Difuso , Adulto , Humanos , Anciano , Estudios Prospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Estudios Retrospectivos , Rituximab/uso terapéutico , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Ciclofosfamida/uso terapéutico , Prednisona/uso terapéutico , Vincristina/uso terapéutico , Doxorrubicina/uso terapéuticoRESUMEN
Metazoan promoters are enriched in secondary DNA structure-forming motifs, such as G-quadruplexes (G4s). Here we describe 'G4access', an approach to isolate and sequence G4s associated with open chromatin via nuclease digestion. G4access is antibody- and crosslinking-independent and enriches for computationally predicted G4s (pG4s), most of which are confirmed in vitro. Using G4access in human and mouse cells, we identify cell-type-specific G4 enrichment correlated with nucleosome exclusion and promoter transcription. G4access allows measurement of variations in G4 repertoire usage following G4 ligand treatment, HDAC and G4 helicases inhibitors. Applying G4access to cells from reciprocal hybrid mouse crosses suggests a role for G4s in the control of active imprinting regions. Consistently, we also observed that G4access peaks are unmethylated, while methylation at pG4s correlates with nucleosome repositioning on DNA. Overall, our study provides a new tool for studying G4s in cellular dynamics and highlights their association with open chromatin, transcription and their antagonism to DNA methylation.
Asunto(s)
Cromatina , G-Cuádruplex , Animales , Humanos , Ratones , Cromatina/genética , Nucleosomas/genética , ADN/genética , Regiones Promotoras GenéticasRESUMEN
The exploration of heath data by clustering algorithms allows to better describe the populations of interest by seeking the sub-profiles that compose it. This therefore reinforces medical knowledge, whether it is about a disease or a targeted population in real life. Nevertheless, contrary to the so-called conventional biostatistical methods where numerous guidelines exist, the standardization of data science approaches in clinical research remains a little discussed subject. This results in a significant variability in the execution of data science projects, whether in terms of algorithms used, reliability and credibility of the designed approach. Taking the path of parsimonious and judicious choice of both algorithms and implementations at each stage, this article proposes Qluster, a practical workflow for performing clustering tasks. Indeed, this workflow makes a compromise between (1) genericity of applications (e.g. usable on small or big data, on continuous, categorical or mixed variables, on database of high-dimensionality or not), (2) ease of implementation (need for few packages, few algorithms, few parameters, ...), and (3) robustness (e.g. use of proven algorithms and robust packages, evaluation of the stability of clusters, management of noise and multicollinearity). This workflow can be easily automated and/or routinely applied on a wide range of clustering projects. It can be useful both for data scientists with little experience in the field to make data clustering easier and more robust, and for more experienced data scientists who are looking for a straightforward and reliable solution to routinely perform preliminary data mining. A synthesis of the literature on data clustering as well as the scientific rationale supporting the proposed workflow is also provided. Finally, a detailed application of the workflow on a concrete use case is provided, along with a practical discussion for data scientists. An implementation on the Dataiku platform is available upon request to the authors.
RESUMEN
MNase-Seq is a genome-wide procedure that allows mapping of DNA associated to nucleosomes following micrococcal nuclease digestion. It is a rapid and robust technology useful for the analysis of chromatin properties genome-wide at the resolution of mono-nucleosomes. Here, we describe how to produce high-resolution nucleosome maps of cells grown in suspension or adherent mammalian cells. After only three steps: nuclei or cell preparation, native MNase digestion and DNA purification, libraries for high-throughput sequencing can be prepared. Genome-wide nucleosome maps allow analyzing chromatin opening at promoters or enhancers, nucleosome displacement, or labile nucleosome occupancy depending on the digestion condition used. As presented, MNase-Seq is a versatile tool for investigating chromatin dynamics, regulation, and to define open chromatin regions of regulatory elements in mammalian genomes.
Asunto(s)
Elementos de Facilitación Genéticos , Estudio de Asociación del Genoma Completo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Animales , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Mapeo Cromosómico , Biología Computacional/métodos , Biblioteca de GenesRESUMEN
Addressing the heterogeneity of both the outcome of a disease and the treatment response to an intervention is a mandatory pathway for regulatory approval of medicines. In randomized clinical trials (RCTs), confirmatory subgroup analyses focus on the assessment of drugs in predefined subgroups, while exploratory ones allow a posteriori the identification of subsets of patients who respond differently. Within the latter area, subgroup discovery (SD) data mining approach is widely used-particularly in precision medicine-to evaluate treatment effect across different groups of patients from various data sources (be it from clinical trials or real-world data). However, both the limited consideration by standard SD algorithms of recommended criteria to define credible subgroups and the lack of statistical power of the findings after correcting for multiple testing hinder the generation of hypothesis and their acceptance by healthcare authorities and practitioners. In this paper, we present the Q-Finder algorithm that aims to generate statistically credible subgroups to answer clinical questions, such as finding drivers of natural disease progression or treatment response. It combines an exhaustive search with a cascade of filters based on metrics assessing key credibility criteria, including relative risk reduction assessment, adjustment on confounding factors, individual feature's contribution to the subgroup's effect, interaction tests for assessing between-subgroup treatment effect interactions and tests adjustment (multiple testing). This allows Q-Finder to directly target and assess subgroups on recommended credibility criteria. The top-k credible subgroups are then selected, while accounting for subgroups' diversity and, possibly, clinical relevance. Those subgroups are tested on independent data to assess their consistency across databases, while preserving statistical power by limiting the number of tests. To illustrate this algorithm, we applied it on the database of the International Diabetes Management Practice Study (IDMPS) to better understand the drivers of improved glycemic control and rate of episodes of hypoglycemia in type 2 diabetics patients. We compared Q-Finder with state-of-the-art approaches from both Subgroup Identification and Knowledge Discovery in Databases literature. The results demonstrate its ability to identify and support a short list of highly credible and diverse data-driven subgroups for both prognostic and predictive tasks.
RESUMEN
Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds DNA at CArG (CC[A/T]6GG) domains in association with myocardin-family proteins (eg, myocardin-related transcription factor A [MRTFA]) or the ternary complex factor family of E26 transformation-specific (ETS) proteins. In primary hematopoietic cells, knockout of either SRF or MRTFA decreases megakaryocyte (Mk) maturation causing thrombocytopenia. The human erythroleukemia (HEL) cell line mimics the effects of MRTFA on Mk maturation, and MRTFA overexpression (MRTFAOE) in HEL cells enhances megakaryopoiesis. To identify the mechanisms underlying these effects, we performed integrated analyses of anti-SRF chromatin immunoprecipitation (ChIP) and RNA-sequencing data from noninduced and phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])-induced HEL cells, with and without MRTFAOE We found that 11% of genes were upregulated with TPA induction, which was enhanced by MRTFAOE, resulting in an upregulation of 25% of genes. MRTFAOE increased binding of SRF to genomic sites and enhanced TPA-induced expression of SRF target genes. The TPA-induced genes are predicted to be regulated by SRF and ETS factors, whereas those upregulated by TPA plus MRTFAOE lack ETS binding motifs, and MRTFAOE skews SRF binding to genomic regions with CArG sites in regions relatively lacking in ETS binding motifs. Finally, ChIP-polymerase chain reaction using HEL cells and primary human CD34+ cell-derived subpopulations confirms that both SRF and MRTFA have increased binding during megakaryopoiesis at upregulated target genes (eg, CORO1A). We show for the first time that MRTFA increases both the genomic association and activity of SRF and upregulates genes that enhance primary human megakaryopoiesis.
Asunto(s)
Megacariocitos/metabolismo , Factor de Respuesta Sérica/genética , Transactivadores/genética , Humanos , Transactivadores/metabolismoRESUMEN
The transcription initiation and elongation steps of protein-coding genes usually rely on unrelated protein complexes. However, the TFIIS elongation factor is implicated in both processes. We found that, in the absence of the Med31 Mediator subunit, yeast cells required the TFIIS polymerase II (Pol II)-binding domain but not its RNA cleavage stimulatory activity that is associated with its elongation function. We also found that the TFIIS Pol II-interacting domain was needed for the full recruitment of Pol II to several promoters in the absence of Med31. This work demonstrated that, in addition to its thoroughly characterized role in transcription elongation, TFIIS is implicated through its Pol II-binding domain in the formation or stabilization of the transcription initiation complex in vivo.