RESUMO
The transition of oxidized 5-methylcytosine (5mC) intermediates into the base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. We report here that UHRF2, the only paralog of UHRF1 in mammals that fails to rescue Uhrf1-/- phenotype, is physically and functionally associated with BER complex. We show that UHRF2 is allosterically activated by 5-hydroxymethylcytosine (5hmC) and acts as a ubiquitin E3 ligase to catalyze K33-linked polyubiquitination of XRCC1. This nonproteolytic action stimulates XRCC1's interaction with the ubiquitin binding domain-bearing RAD23B, leading to the incorporation of TDG into BER complex. Integrative epigenomic analysis in mouse embryonic stem cells reveals that Uhrf2-fostered TDG-RAD23B-BER complex is functionally linked to the completion of DNA demethylation at active promoters and that Uhrf2 ablation impedes DNA demethylation on latent enhancers that undergo poised-to-active transition during neuronal commitment. Together, these observations highlight an essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation.
Assuntos
5-Metilcitosina/análogos & derivados , Regulação Alostérica/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Desmetilação do DNA , Metilação de DNA/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Ligação Proteica/genéticaRESUMO
The de novo assembly and post-splicing reassembly of the U4/U6.U5 tri-snRNP remain to be investigated. We report here that ZIP, a protein containing a CCCH-type zinc finger and a G-patch domain, as characterized by us previously, regulates pre-mRNA splicing independent of RNA binding. We found that ZIP physically associates with the U4/U6.U5 tri-small nuclear ribonucleoprotein (tri-snRNP). Remarkably, the ZIP-containing tri-snRNP, which has a sedimentation coefficient of â¼35S, is a tri-snRNP that has not been described previously. We also found that the 35S tri-snRNP contains hPrp24, indicative of a state in which the U4/U6 di-snRNP is integrating with the U5 snRNP. We found that the 35S tri-snRNP is enriched in the Cajal body, indicating that it is an assembly intermediate during 25S tri-snRNP maturation. We showed that the 35S tri-snRNP also contains hPrp43, in which ATPase/RNA helicase activities are stimulated by ZIP. Our study identified, for the first time, a tri-snRNP intermediate, shedding new light on the de novo assembly and recycling of the U4/U6.U5 tri-snRNP.
Assuntos
Processamento Alternativo , Antígenos de Neoplasias/metabolismo , Biogênese de Organelas , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Spliceossomos/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Corpos Enovelados/química , Corpos Enovelados/enzimologia , Corpos Enovelados/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Células MCF-7 , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Peso Molecular , Mutação , Coloração Negativa , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Multimerização Proteica , Estabilidade Proteica , RNA Helicases/química , RNA Helicases/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Spliceossomos/química , Spliceossomos/enzimologia , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genéticaRESUMO
EGFR is required for animal development, and dysregulation of EGFR is critically implicated in malignant transformation. However, the molecular mechanism underlying the regulation of EGFR expression remains poorly explored. Here we report that the zinc-finger protein ZNF516 is a transcription repressor. ZNF516 is physically associated with the CtBP/LSD1/CoREST complex and transcriptionally represses a cohort of genes including EGFR that are critically involved in cell proliferation and motility. We demonstrate that the ZNF516-CtBP/LSD1/CoREST complex inhibits the proliferation and invasion of breast cancer cells in vitro and suppresses breast cancer growth and metastasis in vivo. Significantly, low expression of ZNF516 is positively associated with advanced pathological staging and poor survival of breast carcinomas. Our data indicate that ZNF516 is a transcription repressor and a potential suppressor of EGFR, adding to the understanding of EGFR-related breast carcinogenesis and supporting the pursuit of ZNF516 as a potential therapeutic target for breast cancer. EGFR is a well-known oncogene; however, the mechanisms regulating its expression are still unclear. Here, analysing genome-wide chromatin associations, the authors show that in breast cancer cells ZNF516 represses EGFR transcription through the interaction with the CtBP/LSD1/CoREST complex.
Assuntos
Neoplasias da Mama/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/fisiologia , Receptores ErbB/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/metabolismo , Feminino , Células HEK293 , Histona Desmetilases/metabolismo , Humanos , Estimativa de Kaplan-Meier , Modelos Genéticos , Invasividade Neoplásica/genética , Proteínas do Tecido Nervoso/metabolismo , Transporte ProteicoRESUMO
The pathophysiological function of the forkhead transcription factor FOXN3 remains to be explored. Here we report that FOXN3 is a transcriptional repressor that is physically associated with the SIN3A repressor complex in estrogen receptor-positive (ER+) cells. RNA immunoprecipitation-coupled high-throughput sequencing identified that NEAT1, an estrogen-inducible long noncoding RNA, is required for FOXN3 interactions with the SIN3A complex. ChIP-Seq and deep sequencing of RNA genomic targets revealed that the FOXN3-NEAT1-SIN3A complex represses genes including GATA3 that are critically involved in epithelial-to-mesenchymal transition (EMT). We demonstrated that the FOXN3-NEAT1-SIN3A complex promotes EMT and invasion of breast cancer cells in vitro as well as dissemination and metastasis of breast cancer in vivo. Interestingly, the FOXN3-NEAT1-SIN3A complex transrepresses ER itself, forming a negative-feedback loop in transcription regulation. Elevation of both FOXN3 and NEAT1 expression during breast cancer progression corresponded to diminished GATA3 expression, and high levels of FOXN3 and NEAT1 strongly correlated with higher histological grades and poor prognosis. Our experiments uncovered that NEAT1 is a facultative component of the SIN3A complex, shedding light on the mechanistic actions of NEAT1 and the SIN3A complex. Further, our study identified the ERα-NEAT1-FOXN3/NEAT1/SIN3A-GATA3 axis that is implicated in breast cancer metastasis, providing a mechanistic insight into the pathophysiological function of FOXN3.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transição Epitelial-Mesenquimal , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias da Mama/genética , Carcinoma/metabolismo , Proteínas de Ciclo Celular/genética , Progressão da Doença , Receptor alfa de Estrogênio/metabolismo , Feminino , Fatores de Transcrição Forkhead , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Hormônios/metabolismo , Humanos , Células MCF-7 , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição GênicaRESUMO
FOXA1 functions in epigenetic reprogramming and is described as a 'pioneer factor'. However, exactly how FOXA1 achieves these remarkable biological functions is not fully understood. Here we report that FOXA1 associates with DNA repair complexes and is required for genomic targeting of DNA polymerase ß (POLB) in human cells. Genome-wide DNA methylomes demonstrate that the FOXA1 DNA repair complex is functionally linked to DNA demethylation in a lineage-specific fashion. Depletion of FOXA1 results in localized reestablishment of methylation in a large portion of FOXA1-bound regions, and the regions with the most consistent hypermethylation exhibit the greatest loss of POLB and are represented by active promoters and enhancers. Consistently, overexpression of FOXA1 commits its binding sites to active DNA demethylation in a POLB-dependent manner. Finally, FOXA1-associated DNA demethylation is tightly coupled with estrogen receptor genomic targeting and estrogen responsiveness. Together, these results link FOXA1-associated DNA demethylation to transcriptional pioneering by FOXA1.