RESUMO
The nuclear protein CCCTC-binding factor (CTCF) has diverse roles in chromatin architecture and gene regulation. Functionally, CTCF associates with thousands of genomic sites and interacts with proteins, such as cohesin, or non-coding RNAs to facilitate specific transcriptional programming. In this study, we examined CTCF during the cellular stress response in human primary cells using immune-blotting, quantitative real time-PCR, chromatin immunoprecipitation-sequence (ChIP-seq) analysis, mass spectrometry, RNA immunoprecipitation-sequence analysis (RIP-seq), and Airyscan confocal microscopy. Unexpectedly, we found that CTCF is exquisitely sensitive to diverse forms of stress in normal patient-derived human mammary epithelial cells (HMECs). In HMECs, a subset of CTCF protein forms complexes that localize to Serine/arginine-rich splicing factor (SC-35)-containing nuclear speckles. Upon stress, this species of CTCF protein is rapidly downregulated by changes in protein stability, resulting in loss of CTCF from SC-35 nuclear speckles and changes in CTCF-RNA interactions. Our ChIP-seq analysis indicated that CTCF binding to genomic DNA is largely unchanged. Restoration of the stress-sensitive pool of CTCF protein abundance and re-localization to nuclear speckles can be achieved by inhibition of proteasome-mediated degradation. Surprisingly, we observed the same characteristics of the stress response during neuronal differentiation of human pluripotent stem cells (hPSCs). CTCF forms stress-sensitive complexes that localize to SC-35 nuclear speckles during a specific stage of neuronal commitment/development but not in differentiated neurons. We speculate that these particular CTCF complexes serve a role in RNA processing that may be intimately linked with specific genes in the vicinity of nuclear speckles, potentially to maintain cells in a certain differentiation state, that is dynamically regulated by environmental signals. The stress-regulated activity of CTCF is uncoupled in persistently stressed, epigenetically re-programmed "variant" HMECs and certain cancer cell lines. These results reveal new insights into CTCF function in cell differentiation and the stress-response with implications for oxidative damage-induced cancer initiation and neuro-degenerative diseases.
Assuntos
Fator de Ligação a CCCTC/genética , Proteínas de Ligação a DNA/genética , Neoplasias/genética , Doenças Neurodegenerativas/genética , Fatores de Processamento de Serina-Arginina/genética , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Cromatina , Cromossomos , Epigênese Genética/genética , Regulação da Expressão Gênica , Genômica , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Ligação Proteica , Processamento Pós-Transcricional do RNA/genética , Estresse Fisiológico/genéticaRESUMO
Cellular stress results in profound changes in RNA and protein synthesis. How cells integrate this intrinsic, p53-centered program with extracellular signals is largely unknown. We demonstrate that TGF-ß1 signaling interferes with the stress response through coordinate transcriptional and translational repression of p53 levels, which reduces p53-activated transcription, and apoptosis in precancerous cells. Mechanistically, E2F-4 binds constitutively to the TP53 gene and induces transcription. TGF-ß1-activated Smads are recruited to a composite Smad/E2F-4 element by an E2F-4/p107 complex that switches to a Smad corepressor, which represses TP53 transcription. TGF-ß1 also causes dissociation of ribosomal protein RPL26 and elongation factor eEF1A from p53 mRNA, thereby reducing p53 mRNA association with polyribosomes and p53 translation. TGF-ß1 signaling is dominant over stress-induced transcription and translation of p53 and prevents stress-imposed downregulation of Smad proteins. Thus, crosstalk between the TGF-ß and p53 pathways defines a major node of regulation in the cellular stress response, enhancing drug resistance.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Fator de Transcrição E2F4/genética , Fator de Transcrição E2F4/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/citologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Estresse Fisiológico/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation.
Assuntos
Epigênese Genética , Genoma Humano , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Fatores de Transcrição/fisiologia , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , FosforilaçãoRESUMO
p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, we examined the role of core promoter structures in this process. We find that the p21 core promoter directs rapid, TATA box-dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. We define a downstream element within the Fas/APO1 core promoter that is essential for its activation, and identify nuclear transcription factor Y (NF-Y) as its binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Fator de Ligação a CCAAT/metabolismo , Células HeLa , Humanos , Cinética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão , Estresse Fisiológico/genética , TATA Box/genética , Receptor fas/genética , Receptor fas/metabolismoRESUMO
The p16(INK4a) tumor suppressor gene is a frequent target of epigenetic inactivation in human cancers, which is an early event in breast carcinogenesis. We describe the existence of a chromatin boundary upstream of the p16 gene that is lost when this gene is aberrantly silenced. We show that the multifunctional protein CTCF associates in the vicinity of this boundary and absence of binding strongly coincides with p16 silencing in multiple types of cancer cells. CTCF binding also correlates with RASSF1A and CDH1 gene activation, and CTCF interaction is absent when these genes are methylated and silenced. Interestingly, defective poly(ADP-ribosyl)ation of CTCF and dissociation from the molecular chaperone Nucleolin occur in p16-silenced cells, abrogating its proper function. Thus, destabilization of specific chromosomal boundaries through aberrant crosstalk between CTCF, poly(ADP-ribosyl)ation, and DNA methylation may be a general mechanism to inactivate tumor suppressor genes and initiate tumorigenesis in numerous forms of human cancers.
Assuntos
Neoplasias da Mama/metabolismo , Cromatina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Inativação Gênica , Proteínas Repressoras/metabolismo , Antígenos CD , Neoplasias da Mama/genética , Fator de Ligação a CCCTC , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica , Humanos , Modelos Genéticos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drug-tolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Paclitaxel/farmacologia , RNA Neoplásico , Análise de Sequência de RNA , Transcrição Gênica , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Fundamental mechanisms that regulate chromatin assembly and transcription have been elucidated recently using genetics and highly defined biochemical systems. Once DNA is packaged into chromatin, its function is controlled by the ordered recruitment of diverse enzymatic complexes that structurally remodel or chemically modify nucleosomes. Recent studies provide insight into the functional selectivity of chromatin-remodeling and -modifying complexes and how they act in specific combinations to regulate individual genes and cellular pathways.
Assuntos
Cromatina/metabolismo , Transcrição Gênica , Acetiltransferases/metabolismo , Animais , Inativação Gênica , Histona Acetiltransferases , Cinética , Substâncias Macromoleculares , Modelos Genéticos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Ativação TranscricionalRESUMO
Chromatin-remodeling complexes are biochemically diverse, functionally selective machines that regulate crucial aspects of DNA metabolism, including transcription and chromatin assembly. These complexes modulate histone-DNA interactions to affect nucleosome repositioning and disassembly, and histone variant exchange, thereby generating compositionally specialized chromatin. Recent studies have revealed precise mechanisms by which specific remodelers control the transition from proliferating progenitors to committed cells through a highly synchronized switch in transcriptional programs. This involves temporal and, often, signal-dependent gene-targeted interactions between individual remodelers and tissue-specific master proteins that regulate myogenesis, neurogenesis and lymphogenesis. Distinct remodelers have also been shown to direct self-renewal of different types of stem cells in response to particular microenvironments.
Assuntos
Diferenciação Celular , Montagem e Desmontagem da Cromatina/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Desenvolvimento Muscular , Neurônios/citologia , Neurônios/metabolismo , Transdução de SinaisRESUMO
Poly(ADP)ribosylation (PARylation) of the chromatin architectural protein CTCF is critical for CTCF-dependent regulation of chromatin boundary and insulator elements. Loss of CTCF PARylation results in epigenetic silencing of certain tumor suppressor genes through destabilization of nearby chromatin boundaries. We investigated the metabolic and mechanistic processes that regulate PARP-1-mediated CTCF PARylation in human cancer cell lines and discovered a key role for the expression and activity of ß-NAD+ salvage enzymes, NAMPT and NMNAT-1. These enzymes are downregulated in cells that exhibit reduced CTCF PARylation, resulting in a decreased concentration of nuclear ß-NAD+. In these cells, decreased NMNAT-1 expression is enforced by a proteasome-mediated feedback loop resulting in degradation of NMNAT-1, transcriptional repression of NAMPT, and suppression of PARP-1 activity. Interestingly, dePARylated CTCF is associated in a stable protein complex with PARP-1 and NMNAT-1 in cancer cells harboring silenced tumor suppressor genes. Although the metabolic context in these cells favors suppression of PARP-1 activity, CTCF PARylation can be restored by Protein Kinase C (PKC) signaling. PKC induces dissociation of the catalytically inactive PARP-1/NMNAT-1/CTCF protein complex and phosphorylation of NMNAT-1, which stimulates its proteasome-mediated degradation. Our findings suggest that CTCF PARylation is underpinned by a cellular metabolic context engendered by regulation of the ß-NAD+ salvage pathway in which NMNAT-1 acts as a rheostat to control localized ß-NAD+ synthesis at CTCF/PARP-1 complexes.
RESUMO
The HS2 enhancer in the beta-globin locus control region (LCR) regulates transcription of the globin genes 10-50 kb away. Earlier studies show that a transcription mechanism initiated by the HS2 enhancer through the intervening DNA in the direction of the cis-linked promoter and gene mediates long-range enhancer function. Here, we further analyzed the enhancer-initiated RNAs and their mode of transcription from the HS2 enhancer in the endogenous genome of erythroid K562 cells, in plasmids integrated into K562 cells and in purified DNA used as template in in vitro transcription reactions. We found that the HS2 enhancer was able to initiate transcription autonomously in the absence of a cis-linked globin promoter. The enhancer-initiated, intergenic RNAs were different from the mRNA synthesized at the promoter in several aspects. The enhancer RNAs were synthesized not from a defined site but from multiple sites both within and as far as 1 kb downstream of the enhancer. The enhancer RNAs did not appear to contain a normal cap structure at the 5' ends. They were polyadenylated at multiple sites within 3 kb downstream of their initiation sites and were therefore shorter than 3 kb in lengths. The enhancer RNAs remained in discrete spots within the nucleus and were not processed into mRNA or translated into proteins. These particular features of enhancer-initiated transcription indicate that the transcriptional complex assembled by the enhancer was different from the basal transcription complex assembled at the promoter. The results suggest that in synthesizing non-coding, intergenic RNAs, the enhancer-assembled transcription complex could track through the intervening DNA to reach the basal promoter complex and activate efficient mRNA synthesis from the promoter.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Globinas/genética , RNA Mensageiro/biossíntese , Transcrição Gênica/genética , DNA Intergênico , Regulação da Expressão Gênica/genética , Humanos , Substâncias Macromoleculares , Regiões Promotoras Genéticas/genéticaRESUMO
Deregulated expression of COX-2 has been causally linked to development, progression, and outcome of several types of human cancer. We describe a novel fundamental level of transcriptional control of COX-2 expression. Using primary human mammary epithelial cells and monocyte/macrophage cell lines, we show that the chromatin boundary/insulator factor CTCF establishes an open chromatin domain and induces expression of a long non-coding RNA within the upstream promoter region of COX-2. Upon induction of COX-2 expression, the lncRNA associates with p50, a repressive subunit of NF-κB, and occludes it from the COX-2 promoter, potentially facilitating interaction with activation-competent NF-κB p65/p50 dimers. This enables recruitment of the p300 histone acetyltransferase, a domain-wide increase in histone acetylation and assembly of RNA Polymerase II initiation complexes. Our findings reveal an unexpected mechanism of gene control by lncRNA-mediated repressor occlusion and identify the COX-2-lncRNA, PACER, as a new potential target for COX-2-modulation in inflammation and cancer.DOI: http://dx.doi.org/10.7554/eLife.01776.001.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Monócitos/fisiologia , Subunidade p50 de NF-kappa B/antagonistas & inibidores , RNA não Traduzido/metabolismo , Transcrição Gênica , Células Cultivadas , Ciclo-Oxigenase 2/genética , HumanosRESUMO
The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes, including NANOG, SOX2, cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sítios de Ligação , Biomarcadores/metabolismo , Fator de Ligação a CCCTC , Diferenciação Celular/genética , Linhagem Celular , Proteínas Cromossômicas não Histona , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Loci Gênicos/genética , Fator 3 de Diferenciação de Crescimento/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Biológicos , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Ligação Proteica/genética , Proteínas/metabolismoRESUMO
Our current understanding of eukaryotic transcription has greatly benefited from use of small molecule inhibitors that have delineated multiple regulatory steps in site-specific initiation and elongation of RNA synthesis by multiple forms of RNA polymerase (RNAP). This class of "transcription" drugs is also of therapeutic interest and under evaluation in clinical trials. However, to date very few small molecules that directly abolish transcription have been identified, particularly those that act at the level of RNAP II initiation. Using a biochemical assay that measures transcription from recombinant, natural p53-responsive promoters and an artificial "super" promoter, we have identified three distinct small molecules that inhibit mRNA synthesis in vitro. Unexpectedly, these are kinase inhibitors, Hypericin, Rottlerin, and SP600125, with known substrates, which we find also strongly impair transcriptional initiation (IC50s = µM range) by targeting specific components of the RNAP II pre-initiation complex. When measured before and during transcription in vitro, one common target of inhibition by all three compounds is modification of the TATA Binding Protein (TBP) within the RNAP II holocomplex as it converts to an active transcribing enzyme. On this basis, by blocking the critical step of TBP modification, transcriptional initiation is effectively abolished even on structurally distinct core promoters.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , RNA Polimerase II/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteína de Ligação a TATA-Box/antagonistas & inibidores , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Cancer cells rapidly evolve drug resistance through somatic evolution and, in order to continue growth in the metastatic phase, violate the organism-wide consensus of regulated growth and beneficial communal interactions. We suggest that there is a fundamental mechanistic connection between the rapid evolution of resistance to chemotherapy in cellular communities within malignant tissues and the rapid evolution of antibiotic resistance in bacterial communities. We propose that this evolution is the result of a programmed and collective stress response performed by interacting cells, and that, given this fundamental connection, studying bacterial communities can provide deeper insights into the dynamics of adaptation and the evolution of cells within tumours.
Assuntos
Antineoplásicos/uso terapêutico , Farmacorresistência Bacteriana/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Humanos , Modelos Biológicos , Neoplasias/etiologiaRESUMO
The composition of chromatin-remodeling complexes dictates how these enzymes control transcriptional programs and cellular identity. In the present study we investigated the composition of SWI/SNF complexes in embryonic stem cells (ESCs). In contrast to differentiated cells, ESCs have a biased incorporation of certain paralogous SWI/SNF subunits with low levels of BRM, BAF170, and ARID1B. Upon differentiation, the expression of these subunits increases, resulting in a higher diversity of compositionally distinct SWI/SNF enzymes. We also identified BRD7 as a novel component of the Polybromo-associated BRG1-associated factor (PBAF) complex in both ESCs and differentiated cells. Using short hairpin RNA-mediated depletion of BRG1, we showed that SWI/SNF can function as both a repressor and an activator in pluripotent cells, regulating expression of developmental modifiers and signaling components such as Nodal, ADAMTS1, BMI-1, CRABP1, and thyroid releasing hormone. Knockdown studies of PBAF-specific BRD7 and of a signature subunit within the BAF complex, ARID1A, showed that these two subcomplexes affect SWI/SNF target genes differentially, in some cases even antagonistically. This may be due to their different biochemical properties. Finally we examined the role of SWI/SNF in regulating its target genes during differentiation. We found that SWI/SNF affects recruitment of components of the preinitiation complex in a promoter-specific manner to modulate transcription positively or negatively. Taken together, our results provide insight into the function of compositionally diverse SWI/SNF enzymes that underlie their inherent gene-specific mode of action.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , DNA Helicases/metabolismo , Humanos , Camundongos , Modelos Biológicos , Complexos Multiproteicos/química , Proteínas Nucleares/metabolismo , Transcrição GênicaRESUMO
Activation of the p53 pathway mediates cellular responses to diverse forms of stress. Here we report that the p53 target gene p21(CIP1) is regulated by stress at post-initiation steps through conversion of paused RNA polymerase II (RNAP II) into an elongating form. High-resolution chromatin immunoprecipitation assays (ChIP) demonstrate that p53-dependent activation of p21(CIP1) transcription after DNA damage occurs concomitantly with changes in RNAP II phosphorylation status and recruitment of the elongation factors DSIF (DRB Sensitivity-Inducing Factor), P-TEFb (Positive Transcription Elongation Factor b), TFIIH, TFIIF, and FACT (Facilitates Chromatin Transcription) to distinct regions of the p21(CIP1) locus. Paradoxically, pharmacological inhibition of P-TEFb leads to global inhibition of mRNA synthesis but activation of the p53 pathway through p53 accumulation, expression of specific p53 target genes, and p53-dependent apoptosis. ChIP analyses of p21(CIP1) activation in the absence of functional P-TEFb reveals the existence of two distinct kinases that phosphorylate Ser5 of the RNAP II C-terminal domain (CTD). Importantly, CTD phosphorylation at Ser2 is not required for p21(CIP1) transcription, mRNA cleavage, or polyadenylation. Furthermore, recruitment of FACT requires CTD kinases, yet FACT is dispensable for p21(CIP1) expression. Thus, select genes within the p53 pathway bypass the requirement for P-TEFb and RNAP II phosphorylation to trigger a cellular response to inhibition of global mRNA synthesis.
Assuntos
Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p21/química , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/análise , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/farmacologia , Citometria de Fluxo , Células HCT116 , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Cinética , Proteínas Nucleares/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , Fator B de Elongação Transcricional Positiva/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase II/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/química , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy approximately 4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.
Assuntos
Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , DNA/genética , DNA/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , Genoma Humano , Hepatócitos/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Distribuição Tecidual , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
The physiologically coordinated expression of our genome requires exquisite regulation of gene specificity. Recent advances demonstrate that this formidable task is accomplished by diverse mechanisms and networks that operate at distinct levels within the nucleus.
Assuntos
Regulação da Expressão Gênica/fisiologia , Animais , Núcleo Celular/fisiologia , Cromatina/metabolismo , Humanos , Modelos Genéticos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Mammalian SWI/SNF chromatin remodeling complexes are involved in critical aspects of cellular growth and genomic stability. Each complex contains one of two highly homologous ATPases, BRG1 and BRM, yet little is known about their specialized functions. We show that BRG1and BRM associate with different promoters during cellular proliferation and differentiation, and in response to specific signaling pathways by preferential interaction with certain classes of transcription factors. BRG1 binds to zinc finger proteins through a unique N-terminal domain that is not present in BRM. BRM interacts with two ankyrin repeat proteins that are critical components of Notch signal transduction. Thus, BRG1 and BRM complexes may direct distinct cellular processes by recruitment to specific promoters through protein-protein interactions that are unique to each ATPase.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Diferenciação Celular , Divisão Celular , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/genética , DNA Helicases , Humanos , Técnicas In Vitro , Camundongos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica , Dedos de ZincoRESUMO
The tumor suppressor protein p53 regulates transcriptional programs that control the response to cellular stress. We show that distinct mechanisms exist to activate p53 target genes as revealed by marked differences in affinities and damage-specific recruitment of transcription initiation components. p53 functions in a temporal manner to regulate promoter activity both before and after stress. Before DNA damage, basal levels of p53 are required to assemble a poised RNA polymerase II initiation complex on the p21 promoter. RNA pol II is converted into an elongating form shortly after stress but before p53 stabilization. Proapoptotic promoters, such as Fas/APO1, have low levels of bound RNA pol II but undergo damage-induced activation through efficient reinitiation. Surprisingly, in a p53-dependent process key basal factors TAFII250 and TFIIB assemble into the transcription machinery in a stress- and promoter-specific manner, behaving as differential cofactors for p53 action after distinct types of DNA damage.