RESUMO
Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. In vivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.
RESUMO
Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.
Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Hormônios , Transdução de SinaisRESUMO
Androgen receptor (AR) is a main driver for castration-resistant prostate cancer (CRPC). c-Myc is an oncogene underlying prostate tumorigenesis. Here, we find that the deubiquitinase USP11 targets both AR and c-Myc in prostate cancer (PCa). USP11 expression was up-regulated in metastatic PCa and CRPC. USP11 knockdown (KD) significantly inhibited PCa cell growth. Our RNA-seq studies revealed AR and c-Myc as the top transcription factors altered after USP11 KD. ChIP-seq analysis showed that either USP11 KD or replacement of endogenous USP11 with a catalytic-inactive USP11 mutant significantly decreased chromatin binding by AR and c-Myc. We find that USP11 employs two mechanisms to up-regulate AR and c-Myc levels: namely, deubiquitination of AR and c-Myc proteins to increase their stability and deubiquitination of H2A-K119Ub, a repressive histone mark, on promoters of AR and c-Myc genes to increase their transcription. AR and c-Myc reexpression in USP11-KD PCa cells partly rescued cell growth defects. Thus, our studies reveal a tumor-promoting role for USP11 in aggressive PCa through upregulation of AR and c-Myc activities and support USP11 as a potential target against PCa.
Assuntos
Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-myc , Receptores Androgênicos , Tioléster Hidrolases , Humanos , Masculino , Linhagem Celular Tumoral , Proliferação de Células/genética , Histonas/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Ubiquitinação , Regulação para CimaRESUMO
Overexpression of androgen receptor (AR) is the primary cause of castration-resistant prostate cancer, although mechanisms upregulating AR transcription in this context are not well understood. Our RNA-seq studies revealed that SMAD3 knockdown decreased levels of AR and AR target genes, whereas SMAD4 or SMAD2 knockdown had little or no effect. ChIP-seq analysis showed that SMAD3 knockdown decreased global binding of AR to chromatin. Mechanistically, we show that SMAD3 binds to intron 3 of the AR gene to promote AR expression. Targeting these binding sites by CRISPRi reduced transcript levels of AR and AR targets. In addition, â¼50% of AR and SMAD3 ChIP-seq peaks overlapped, and SMAD3 may also cooperate with or co-activate AR for AR target expression. Functionally, AR re-expression in SMAD3-knockdown cells partially rescued AR target expression and cell growth defects. The SMAD3 peak in AR intron 3 overlapped with H3K27ac ChIP-seq and ATAC-seq peaks in datasets of prostate cancer. AR and SMAD3 mRNAs were upregulated in datasets of metastatic prostate cancer and CRPC compared with primary prostate cancer. A SMAD3 PROTAC inhibitor reduced levels of AR, AR-V7 and AR targets in prostate cancer cells. This study suggests that SMAD3 could be targeted to inhibit AR in prostate cancer.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Proteína Smad3 , Humanos , Masculino , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
SARS-CoV-2 entry into cells requires specific host proteases; however, no successful in vivo applications of host protease inhibitors have yet been reported for treatment of SARS-CoV-2 pathogenesis. Here we describe a chemically engineered nanosystem encapsulating CRISPR-Cas13d, developed to specifically target lung protease cathepsin L (Ctsl) messenger RNA to block SARS-CoV-2 infection in mice. We show that this nanosystem decreases lung Ctsl expression in normal mice efficiently, specifically and safely. We further show that this approach extends survival of mice lethally infected with SARS-CoV-2, correlating with decreased lung virus burden, reduced expression of proinflammatory cytokines/chemokines and diminished severity of pulmonary interstitial inflammation. Postinfection treatment by this nanosystem dramatically lowers the lung virus burden and alleviates virus-induced pathological changes. Our results indicate that targeting lung protease mRNA by Cas13d nanosystem represents a unique strategy for controlling SARS-CoV-2 infection and demonstrate that CRISPR can be used as a potential treatment for SARS-CoV-2 infection.
Assuntos
Tratamento Farmacológico da COVID-19 , Animais , Catepsina L , Quimiocinas , Citocinas , Endopeptidases , Pulmão/patologia , Camundongos , Peptídeo Hidrolases , Inibidores de Proteases/farmacologia , RNA Mensageiro/genética , SARS-CoV-2RESUMO
The evolution of prostate cancer from an androgen-dependent state to one that is androgen-independent marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in androgen-independent cancers is poorly understood. We have defined the direct AR-dependent target genes in both androgen-dependent and -independent cancer cells by generating AR-dependent gene expression profiles and AR cistromes. In contrast to what is found in androgen-dependent cells, AR selectively upregulates M-phase cell-cycle genes in androgen-independent cells, including UBE2C, a gene that inactivates the M-phase checkpoint. We find that epigenetic marks at the UBE2C enhancer, notably histone H3K4 methylation and FoxA1 transcription factor binding, are present in androgen-independent cells and direct AR-enhancer binding and UBE2C activation. Thus, the role of AR in androgen-independent cancer cells is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Androgênios/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Histonas/metabolismo , Humanos , Masculino , Neoplasias da Próstata/genética , Ativação Transcricional , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Mediator activates RNA polymerase II (Pol II) function during transcription, but it remains unclear whether Mediator is able to travel with Pol II and regulate Pol II transcription beyond the initiation and early elongation steps. By using in vitro and in vivo transcription recycling assays, we find that human Mediator 1 (MED1), when phosphorylated at the mammal-specific threonine 1032 by cyclin-dependent kinase 9 (CDK9), dynamically moves along with Pol II throughout the transcribed genes to drive Pol II recycling after the initial round of transcription. Mechanistically, MED31 mediates the recycling of phosphorylated MED1 and Pol II, enhancing mRNA output during the transcription recycling process. Importantly, MED1 phosphorylation increases during prostate cancer progression to the lethal phase, and pharmacological inhibition of CDK9 decreases prostate tumor growth by decreasing MED1 phosphorylation and Pol II recycling. Our results reveal a novel role of MED1 in Pol II transcription and identify phosphorylated MED1 as a targetable driver of dysregulated Pol II recycling in cancer.
Assuntos
Neoplasias , RNA Polimerase II , Animais , Humanos , Masculino , Mamíferos/genética , Complexo Mediador/metabolismo , Subunidade 1 do Complexo Mediador/genética , Neoplasias/genética , Fosforilação , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to hormonal therapy resistance and disease progression in prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) up-regulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), an androgen-independent GLS1 isoform with more potent enzymatic activity, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyperproliferation, and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GAC shows better treatment effect for castration-resistant PCa than for hormone-sensitive PCa in vitro and in vivo. In summary, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is one of the key mechanisms in therapeutic resistance and disease progression.
Assuntos
Antagonistas de Androgênios/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Glutaminase/genética , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/uso terapêutico , Animais , Linhagem Celular Tumoral , Biologia Computacional , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutaminase/metabolismo , Glutamina/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Complex organisms require tissue-specific transcriptional programs, yet little is known about how these are established. The transcription factor FoxA1 is thought to contribute to gene regulation through its ability to act as a pioneer factor binding to nucleosomal DNA. Through genome-wide positional analyses, we demonstrate that FoxA1 cell type-specific functions rely primarily on differential recruitment to chromatin predominantly at distant enhancers rather than proximal promoters. This differential recruitment leads to cell type-specific changes in chromatin structure and functional collaboration with lineage-specific transcription factors. Despite the ability of FoxA1 to bind nucleosomes, its differential binding to chromatin sites is dependent on the distribution of histone H3 lysine 4 dimethylation. Together, our results suggest that methylation of histone H3 lysine 4 is part of the epigenetic signature that defines lineage-specific FoxA1 recruitment sites in chromatin. FoxA1 translates this epigenetic signature into changes in chromatin structure thereby establishing lineage-specific transcriptional enhancers and programs.
Assuntos
Elementos Facilitadores Genéticos , Epigênese Genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Genoma Humano , Código das Histonas , Histonas/metabolismo , Humanos , Metilação , Receptores Androgênicos/metabolismoRESUMO
Enzalutamide, a second-generation androgen receptor (AR) antagonist, has demonstrated clinical benefit in men with prostate cancer. However, it only provides a temporary response and modest increase in survival, indicating a rapid evolution of resistance. Previous studies suggest that enzalutamide may function as a partial transcriptional agonist, but the underlying mechanisms for enzalutamide-induced transcription remain poorly understood. Here, we show that enzalutamide stimulates expression of a novel subset of genes distinct from androgen-responsive genes. Treatment of prostate cancer cells with enzalutamide enhances recruitment of pioneer factor GATA2, AR, Mediator subunits MED1 and MED14, and RNA Pol II to regulatory elements of enzalutamide-responsive genes. Mechanistically, GATA2 globally directs enzalutamide-induced transcription by facilitating AR, Mediator and Pol II loading to enzalutamide-responsive gene loci. Importantly, the GATA2 inhibitor K7174 inhibits enzalutamide-induced transcription by decreasing binding of the GATA2/AR/Mediator/Pol II transcriptional complex, contributing to sensitization of prostate cancer cells to enzalutamide treatment. Our findings provide mechanistic insight into the future combination of GATA2 inhibitors and enzalutamide for improved AR-targeted therapy.
Assuntos
Fator de Transcrição GATA2/genética , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/genética , Antagonistas de Receptores de Andrógenos/farmacologia , Benzamidas , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição GATA2/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/genética , Nitrilas , Feniltioidantoína/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Polimerase II/genéticaRESUMO
The constitutively active androgen receptor (AR) splice variant 7 (AR-V7) plays an important role in the progression of castration-resistant prostate cancer (CRPC). Although biomarker studies established the role of AR-V7 in resistance to AR-targeting therapies, how AR-V7 mediates genomic functions in CRPC remains largely unknown. Using a ChIP-exo approach, we show AR-V7 binds to distinct genomic regions and recognizes a full-length androgen-responsive element in CRPC cells and patient tissues. Remarkably, we find dramatic differences in AR-V7 cistromes across diverse CRPC cells and patient tissues, regulating different target gene sets involved in CRPC progression. Surprisingly, we discover that HoxB13 is universally required for and colocalizes with AR-V7 binding to open chromatin across CRPC genomes. HoxB13 pioneers AR-V7 binding through direct physical interaction, and collaborates with AR-V7 to up-regulate target oncogenes. Transcriptional coregulation by HoxB13 and AR-V7 was further supported by their coexpression in tumors and circulating tumor cells from CRPC patients. Importantly, HoxB13 silencing significantly decreases CRPC growth through inhibition of AR-V7 oncogenic function. These results identify HoxB13 as a pivotal upstream regulator of AR-V7-driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7-driven prostate tumors.
Assuntos
Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/biossíntese , Regulação para Cima , Linhagem Celular Tumoral , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Neoplasias de Próstata Resistentes à Castração/genética , Ligação Proteica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Receptores Androgênicos/genéticaRESUMO
Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.
Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Código das Histonas/genética , Histonas/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/deficiência , Neoplasias Hepáticas/genética , Proteínas Supressoras de Tumor/deficiência , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Feminino , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Neoplasias Hepáticas Experimentais/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Terapia de Alvo Molecular , Prognóstico , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Neoplásico/genética , Análise de Sequência de RNA , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human transcription factors recognize specific DNA sequence motifs to regulate transcription. It is unknown whether a single transcription factor is able to bind to distinctly different motifs on chromatin, and if so, what determines the usage of specific motifs. By using a motif-resolution chromatin immunoprecipitation-exonuclease (ChIP-exo) approach, we find that agonist-liganded human androgen receptor (AR) and antagonist-liganded AR bind to two distinctly different motifs, leading to distinct transcriptional outcomes in prostate cancer cells. Further analysis on clinical prostate tissues reveals that the binding of AR to these two distinct motifs is involved in prostate carcinogenesis. Together, these results suggest that unique ligands may switch DNA motifs recognized by ligand-dependent transcription factors in vivo. Our findings also provide a broad mechanistic foundation for understanding ligand-specific induction of gene expression profiles.
Assuntos
Antagonistas de Receptores de Andrógenos/química , Androgênios/química , DNA/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos/metabolismo , Androgênios/metabolismo , Proliferação de Células/fisiologia , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Masculino , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The compaction of nucleosomal structures creates a barrier for DNA-binding transcription factors (TFs) to access their cognate cis-regulatory elements. Pioneer factors (PFs) such as FOXA1 are able to directly access these cis-targets within compact chromatin. However, how these PFs interplay with nucleosomes remains to be elucidated, and is critical for us to understand the underlying mechanism of gene regulation. Here, we have conducted a computational analysis on a strand-specific paired-end ChIP-exo (termed as ChIP-ePENS) data of FOXA1 in LNCaP cells by our novel algorithm ePEST. We find that FOXA1 chromatin binding occurs via four distinct border modes (or footprint boundary patterns), with a preferential footprint boundary patterns relative to FOXA1 motif orientation. In addition, from this analysis three fundamental nucleotide positions (oG, oS and oH) emerged as major determinants for blocking exo-digestion and forming these four distinct border modes. By integrating histone MNase-seq data, we found an astonishingly consistent, 'well-positioned' configuration occurs between FOXA1 motifs and dyads of nucleosomes genome-wide. We further performed ChIP-seq of eight chromatin remodelers and found an increased occupancy of these remodelers on FOXA1 motifs for all four border modes (or footprint boundary patterns), indicating the full occupancy of FOXA1 complex on the three blocking sites (oG, oS and oH) likely produces an active regulatory status with well-positioned phasing for protein binding events. Together, our results suggest a positional-nucleosome-oriented accessing model for PFs seeking target motifs, in which FOXA1 can examine each underlying DNA nucleotide and is able to sense all potential motifs regardless of whether they face inward or outward from histone octamers along the DNA helix axis.
Assuntos
Genoma Humano , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Nucleossomos/metabolismo , Algoritmos , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Biologia Computacional , Histonas/metabolismo , Humanos , Motivos de Nucleotídeos/genética , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each other's binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Fator 3-alfa Nuclear de Hepatócito/fisiologia , Recidiva Local de Neoplasia/metabolismo , Neoplasias da Próstata/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Sítios de Ligação , Biomarcadores Tumorais , Linhagem Celular Tumoral , Sequência Consenso , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Subunidade 1 do Complexo Mediador/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Transcrição GênicaRESUMO
The [A] allele of SNP rs965513 in 9q22 has been consistently shown to be highly associated with increased papillary thyroid cancer (PTC) risk with an odds ratio of â¼1.8 as determined by genome-wide association studies, yet the molecular mechanisms remain poorly understood. Previously, we noted that the expression of two genes in the region, forkhead box E1 (FOXE1) and PTC susceptibility candidate 2 (PTCSC2), is regulated by rs965513 in unaffected thyroid tissue, but the underlying mechanisms were not elucidated. Here, we fine-mapped the 9q22 region in PTC and controls and detected an â¼33-kb linkage disequilibrium block (containing the lead SNP rs965513) that significantly associates with PTC risk. Chromatin characteristics and regulatory element signatures in this block disclosed at least three regulatory elements functioning as enhancers. These enhancers harbor at least four SNPs (rs7864322, rs12352658, rs7847449, and rs10759944) that serve as functional variants. The variant genotypes are associated with differential enhancer activities and/or transcription factor binding activities. Using the chromosome conformation capture methodology, long-range looping interactions of these elements with the promoter region shared by FOXE1 and PTCSC2 in a human papillary thyroid carcinoma cell line (KTC-1) and unaffected thyroid tissue were found. Our results suggest that multiple variants coinherited with the lead SNP and located in long-range enhancers are involved in the transcriptional regulation of FOXE1 and PTCSC2 expression. These results explain the mechanism by which the risk allele of rs965513 predisposes to thyroid cancer.
Assuntos
Carcinoma/genética , Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Neoplasias da Glândula Tireoide/genética , Alelos , Carcinoma Papilar , Linhagem Celular Tumoral , Cromatina/química , Imunoprecipitação da Cromatina , Fatores de Transcrição Forkhead/genética , Predisposição Genética para Doença , Genótipo , Haplótipos , Histonas/química , Humanos , Razão de Chances , Penetrância , Câncer Papilífero da TireoideRESUMO
Alternative splicing (AS), in higher eukaryotes, is one of the mechanisms of post-transcriptional regulation that generate multiple transcripts from the same gene. One particular mode of AS is the skipping event where an exon may be alternatively excluded or constitutively included in the resulting mature mRNA. Both transcript isoforms from this skipping event site, i.e. in which the exon is either included (inclusion isoform) or excluded (skipping isoform), are typically present in one cell, and maintain a subtle balance that is vital to cellular function and dynamics. However, how the prevailing conditions dictate which isoform is expressed and what biological factors might influence the regulation of this process remain areas requiring further exploration. In this study, we have developed a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium and experimentally validated several skipping site predictions by RT-PCR. Furthermore, we integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. Our computational analysis found that splice sites within the skipping-isoform-dominated group (SIDG) tended to exhibit weaker MaxEntScan-calculated splice site strength around middle, 'skipping', exons compared to those in the inclusion-isoform-dominated group (IIDG). We further showed the positional preference pattern of splicing factors, characterized by enrichment in the intronic splice sites immediately bordering middle exons. Finally, our analysis suggested that different epigenetic factors may introduce a variable obstacle in the process of exon-intron boundary establishment leading to skipping events.
Assuntos
Processamento Alternativo , Epigênese Genética , Éxons , Análise de Sequência de RNA , Transcrição Gênica , Sítios de Ligação , Linhagem Celular , Biologia Computacional/métodos , Histonas/metabolismo , Humanos , Células K562 , Motivos de Nucleotídeos , Sítios de Splice de RNA , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In prostate cancer, androgen receptor (AR) binding and androgen-responsive gene expression are defined by hormone-independent binding patterns of the pioneer factors FoxA1 and GATA2. Insufficient evidence of the mechanisms by which GATA2 contributes to this process precludes complete understanding of a key determinant of tissue-specific AR activity. Our observations suggest that GATA2 facilitates androgen-responsive gene expression by three distinct modes of action. By occupying novel binding sites within the AR gene locus, GATA2 positively regulates AR expression before and after androgen stimulation. Additionally, GATA2 engages AR target gene enhancers prior to hormone stimulation, producing an active and accessible chromatin environment via recruitment of the histone acetyltransferase p300. Finally, GATA2 functions in establishing and/or sustaining basal locus looping by recruiting the Mediator subunit MED1 in the absence of androgen. These mechanisms may contribute to the generally positive role of GATA2 in defining AR genome-wide binding patterns that determine androgen-responsive gene expression profiles. We also find that GATA2 and FoxA1 exhibit both independent and codependent co-occupancy of AR target gene enhancers. Identifying these determinants of AR transcriptional activity may provide a foundation for the development of future prostate cancer therapeutics that target pioneer factor function.
Assuntos
Fator de Transcrição GATA2/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Genoma Humano , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genéticaRESUMO
The UBE2C oncogene is overexpressed in many types of solid tumours including the lethal castration-resistant prostate cancer (CRPC). The underlying mechanisms causing UBE2C gene overexpression in CRPC are not fully understood. Here, we show that CRPC-specific enhancers drive UBE2C overexpression in both AR-negative and -positive CRPC cells. We further show that co-activator MED1 recruitment to the UBE2C enhancers is required for long-range UBE2C enhancer/promoter interactions. Importantly, we find that the molecular mechanism underlying MED1-mediated chromatin looping involves PI3K/AKT phosphorylated MED1-mediated recruitment of FoxA1, RNA polymerase II and TATA binding protein and their subsequent interactions at the UBE2C locus. MED1 phosphorylation leads to UBE2C locus looping, UBE2C gene expression and cell growth. Our results not only define a causal role of a post-translational modification (phosphorylation) of a co-activator (MED1) in forming or sustaining an active chromatin structure, but also suggest that development of specific therapies for CRPC should take account of targeting phosphorylated MED1.
Assuntos
Proliferação de Células , Elementos Facilitadores Genéticos/genética , Loci Gênicos/genética , Subunidade 1 do Complexo Mediador/metabolismo , Conformação de Ácido Nucleico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Enzimas de Conjugação de Ubiquitina/genética , Linhagem Celular Tumoral , Cromatina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Subunidade 1 do Complexo Mediador/fisiologia , Fosforilação/genética , Neoplasias da Próstata/mortalidade , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Enzimas de Conjugação de Ubiquitina/biossíntese , Regulação para Cima/genéticaRESUMO
Androgen receptor (AR) is a hormone-activated transcription factor that plays important roles in prostate development and function, as well as malignant transformation. The downstream pathways of AR, however, are incompletely understood. AR has been primarily known as a transcriptional activator inducing prostate-specific gene expression. Through integrative analysis of genome-wide AR occupancy and androgen-regulated gene expression, here we report AR as a globally acting transcriptional repressor. This repression is mediated by androgen-responsive elements (ARE) and dictated by Polycomb group protein EZH2 and repressive chromatin remodeling. In embryonic stem cells, AR-repressed genes are occupied by EZH2 and harbor bivalent H3K4me3 and H3K27me3 modifications that are characteristic of differentiation regulators, the silencing of which maintains the undifferentiated state. Concordantly, these genes are silenced in castration-resistant prostate cancer rendering a stem cell-like lack of differentiation and tumor progression. Collectively, our data reveal an unexpected role of AR as a transcriptional repressor inhibiting non-prostatic differentiation and, upon excessive signaling, resulting in cancerous dedifferentiation.