RESUMO
Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is responsible for the dephosphorylation and inactivation of ERK, JNK and p38, which are mitogen-activated protein kinases involved in cell proliferation, differentiation and apoptosis, but also in inflammation processes. Given its importance for cellular signalling, DUSP4 is subjected to a tight regulation and there is growing evidence that its expression is dysregulated in several tumours. However, the mechanisms underlying DUSP4 transcriptional regulation remain poorly understood. Here, we analysed the regulation of the human DUSP4 promoters 1 and 2, located upstream of exons 1 and 2, respectively, by the cancer-related transcription factors (TFs) STAT3, FOXA1, CTCF and YY1. The presence of binding sites for these TFs was predicted in both promoters through the in silico analysis of DUSP4, and their functionality was assessed through luciferase activity assays. Regulatory activity of the TFs tested was found to be promoter-specific. While CTCF stimulated the activity of promoter 2 that controls the transcription of variants 2 and X1, STAT3 stimulated the activity of promoter 1 that controls the transcription of variant 1. YY1 positively regulated both promoters, although to different extents. Through site-directed mutagenesis, the functionality of YY1 binding sites present in promoter 2 was confirmed. This study provides novel insights into the transcriptional regulation of DUSP4, contributing to a better comprehension of the mechanisms of its dysregulation observed in several types of cancer.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Fosfatases de Especificidade Dupla/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição YY1/metabolismo , Apoptose/fisiologia , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Fosfatases de Especificidade Dupla/metabolismo , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT3/genética , Fator de Transcrição YY1/genéticaRESUMO
The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment.
Assuntos
Eucromatina/metabolismo , Heterocromatina/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Transcrição Gênica , Animais , Fator de Ligação a CCCTC/metabolismo , Metilação de DNA/genética , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Inativação Gênica , Camadas Germinativas/citologia , Histonas/metabolismo , Camundongos , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Longo não Codificante/metabolismo , Células-Tronco/metabolismo , Fator de Transcrição YY1/metabolismoRESUMO
Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (â¼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP-chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Imunidade Adaptativa/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina , Células K562 , Camundongos , Camundongos Knockout , Células NIH 3T3 , Motivos de Nucleotídeos , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/metabolismoRESUMO
The complex life cycle of oncogenic human papillomavirus (HPV) initiates in undifferentiated basal epithelial keratinocytes where expression of the E6 and E7 oncogenes is restricted. Upon epithelial differentiation, E6/E7 transcription is increased through unknown mechanisms to drive cellular proliferation required to support virus replication. We report that the chromatin-organising CCCTC-binding factor (CTCF) promotes the formation of a chromatin loop in the HPV genome that epigenetically represses viral enhancer activity controlling E6/E7 expression. CTCF-dependent looping is dependent on the expression of the CTCF-associated Yin Yang 1 (YY1) transcription factor and polycomb repressor complex (PRC) recruitment, resulting in trimethylation of histone H3 at lysine 27. We show that viral oncogene up-regulation during cellular differentiation results from YY1 down-regulation, disruption of viral genome looping, and a loss of epigenetic repression of viral enhancer activity. Our data therefore reveal a key role for CTCF-YY1-dependent looping in the HPV life cycle and identify a regulatory mechanism that could be disrupted in HPV carcinogenesis.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Papillomaviridae/genética , Fator de Transcrição YY1/metabolismo , Fator de Ligação a CCCTC/genética , Diferenciação Celular/genética , Cromatina/fisiologia , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Epigênese Genética/genética , Histonas/genética , Humanos , Regiões Promotoras Genéticas/genética , Proteínas Repressoras , Fatores de Transcrição , Ativação Transcricional/genética , Replicação Viral/genética , Replicação Viral/fisiologia , Fator de Transcrição YY1/genéticaRESUMO
Hexavalent chromium compounds are well-established respiratory carcinogens used in industrial processes. While inhalation exposure constitutes an occupational risk affecting mostly chromium workers, environmental exposure from drinking water is a widespread gastrointestinal cancer risk, affecting millions of people throughout the world. Cr(VI) is genotoxic, forming protein-Cr-DNA adducts and silencing tumor suppressor genes, but its mechanism of action at the molecular level is poorly understood. Our prior work using FAIRE showed that Cr(VI) disrupted the binding of transcription factors CTCF and AP-1 to their cognate chromatin sites. Here, we used two complementary approaches to test the hypothesis that chromium perturbs chromatin organization and dynamics. DANPOS2 analyses of MNase-seq data identified several chromatin alterations induced by Cr(VI) affecting nucleosome architecture, including occupancy changes at specific genome locations; position shifts of 10 nucleotides or more; and changes in position amplitude or fuzziness. ATAC-seq analysis revealed that Cr(VI) disrupted the accessibility of chromatin regions enriched for CTCF and AP-1 binding motifs, with a significant co-occurrence of binding sites for both factors in the same region. Cr(VI)-enriched CTCF sites were confirmed by ChIP-seq and found to correlate with evolutionarily conserved sites occupied by CTCF in vivo, as determined by comparison with ENCODE-validated CTCF datasets from mouse liver. In addition, more than 30% of the Cr(VI)-enriched CTCF sites were located in promoters of genes differentially expressed from chromium treatment. Our results support the conclusion that Cr(VI) exposure promotes broad changes in chromatin accessibility and suggest that the subsequent effects on transcription regulation may result from disruption of CTCF binding and nucleosome spacing, implicating transcription regulatory mechanisms as primary Cr(VI) targets.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Cromo/efeitos adversos , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise de Sequência de DNA/métodos , Animais , Sítios de Ligação , Linhagem Celular , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Água Potável/efeitos adversos , Água Potável/química , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/química , Fígado/efeitos dos fármacos , Camundongos , Ligação Proteica/efeitos dos fármacosRESUMO
Immunoglobulin heavy-chain (IgH) genes are assembled by DNA rearrangements that juxtapose a variable (VH), a diversity (DH), and a joining (JH) gene segment. Here, we report that in the absence of intergenic control region 1 (IGCR1), the intronic enhancer (Eµ) associates with the next available CTCF binding site located close to VH81X via putative heterotypic interactions involving YY1 and CTCF. The alternate Eµ/VH81X loop leads to formation of a distorted recombination center and altered DH rearrangements and disrupts chromosome conformation that favors distal VH recombination. Cumulatively, these features drive highly skewed, Eµ-dependent recombination of VH81X. Sequential deletion of CTCF binding regions on IGCR1-deleted alleles suggests that they influence recombination of single proximal VH gene segments. Our observations demonstrate that Eµ interacts differently with IGCR1- or VH-associated CTCF binding sites and thereby identify distinct roles for insulator-like elements in directing enhancer activity.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Intergênico/genética , Elementos Facilitadores Genéticos , Genes de Cadeia Pesada de Imunoglobulina , Loci Gênicos , Região Variável de Imunoglobulina/genética , Células Precursoras de Linfócitos B/metabolismo , Recombinação Genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Camundongos da Linhagem 129 , Camundongos Knockout , Conformação de Ácido Nucleico , Células Precursoras de Linfócitos B/imunologia , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.
Assuntos
Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fator de Transcrição YY1/metabolismo , Animais , Fator de Ligação a CCCTC/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , CamundongosRESUMO
CTCF is an architectural protein with a critical role in connecting higher-order chromatin folding in pluripotent stem cells. Recent reports have suggested that CTCF binding is more dynamic during development than previously appreciated. Here, we set out to understand the extent to which shifts in genome-wide CTCF occupancy contribute to the 3D reconfiguration of fine-scale chromatin folding during early neural lineage commitment. Unexpectedly, we observe a sharp decrease in CTCF occupancy during the transition from naïve/primed pluripotency to multipotent primary neural progenitor cells (NPCs). Many pluripotency gene-enhancer interactions are anchored by CTCF, and its occupancy is lost in parallel with loop decommissioning during differentiation. Conversely, CTCF binding sites in NPCs are largely preexisting in pluripotent stem cells. Only a small number of CTCF sites arise de novo in NPCs. We identify another zinc finger protein, Yin Yang 1 (YY1), at the base of looping interactions between NPC-specific genes and enhancers. Putative NPC-specific enhancers exhibit strong YY1 signal when engaged in 3D contacts and negligible YY1 signal when not in loops. Moreover, siRNA knockdown of Yy1 specifically disrupts interactions between key NPC enhancers and their target genes. YY1-mediated interactions between NPC regulatory elements are often nested within constitutive loops anchored by CTCF. Together, our results support a model in which YY1 acts as an architectural protein to connect developmentally regulated looping interactions; the location of YY1-mediated interactions may be demarcated in development by a preexisting topological framework created by constitutive CTCF-mediated interactions.