RESUMO
Androgen receptor (AR) and its splice variants (AR-SVs) promote prostate cancer (PCa) growth by orchestrating transcriptional reprogramming. Mechanisms by which the low complexity and intrinsically disordered primary transactivation domain (AF-1) of AR and AR-SVs regulate transcriptional programming in PCa remains poorly defined. Using omics, live and fixed fluorescent microscopy of cells, and purified AF-1 and AR-V7 recombinant proteins we show here that AF-1 and the AR-V7 splice variant form molecular condensates by liquid-liquid phase separation (LLPS) that exhibit disorder characteristics such as rapid intracellular mobility, coactivator interaction, and euchromatin induction. The LLPS and other disorder characteristics were reversed by a class of small-molecule-selective AR-irreversible covalent antagonists (SARICA) represented herein by UT-143 that covalently and selectively bind to C406 and C327 in the AF-1 region. Interfering with LLPS formation with UT-143 or mutagenesis resulted in chromatin condensation and dissociation of AR-V7 interactome, all culminating in a transcriptionally incompetent complex. Biochemical studies suggest that C327 and C406 in the AF-1 region are critical for condensate formation, AR-V7 function, and UT-143's irreversible AR inhibition. Therapeutically, UT-143 possesses drug-like pharmacokinetics and metabolism properties and inhibits PCa cell proliferation and tumor growth. Our work provides critical information suggesting that clinically important AR-V7 forms transcriptionally competent molecular condensates and covalently engaging C327 and C406 in AF-1, dissolves the condensates, and inhibits its function. The work also identifies a library of AF-1-binding AR and AR-SV-selective covalent inhibitors for the treatment of PCa.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Receptores Androgênicos/metabolismo , Cisteína , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Linhagem Celular Tumoral , Isoformas de Proteínas/metabolismoRESUMO
Evidence mounts, via two studies published in Molecular Cell (Riscal et al., 2016; Wienken et al., 2016), that chromatin-bound MDM2 impacts pluripotency and metabolism to promote survival and proliferation of cancer cells, independently of p53 degradation.
Assuntos
Cromatina , Proteínas Proto-Oncogênicas c-mdm2/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome-wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity.
Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Histonas/metabolismo , Células-Tronco Pluripotentes/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Histona Desacetilases , Histonas/genética , Humanos , Células-Tronco Pluripotentes/citologia , Processamento de Proteína Pós-Traducional/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The methyl-lysine readers plant homeodomain finger protein 20 (PHF20) and its homolog PHF20-like protein 1 (PHF20L1) are known components of the nonspecific lethal (NSL) complex that regulates gene expression through its histone acetyltransferase activity. In the current model, both PHF homologs coexist in the same NSL complex, although this was not formally tested; nor have the functions of PHF20 and PHF20L1 regarding NSL complex integrity and transcriptional regulation been investigated. Here, we perform an in-depth biochemical and functional characterization of PHF20 and PHF20L1 in the context of the NSL complex. Using mass spectrometry, genome-wide chromatin analysis, and protein-domain mapping, we identify the existence of two distinct NSL complexes that exclusively contain either PHF20 or PHF20L1. We show that the C-terminal domains of PHF20 and PHF20L1 are essential for complex formation with NSL, and the Tudor 2 domains are required for chromatin binding. The genome-wide chromatin landscape of PHF20-PHF20L1 shows that these proteins bind mostly to the same genomic regions, at promoters of highly expressed/housekeeping genes. Yet, deletion of PHF20 and PHF20L1 does not abrogate gene expression or impact the recruitment of the NSL complex to those target gene promoters, suggesting the existence of an alternative mechanism that compensates for the transcription of genes whose sustained expression is important for critical cellular functions. This work shifts the current paradigm and lays the foundation for studies on the differential roles of PHF20 and PHF20L1 in regulating NSL complex activity in physiological and diseases states.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Proteínas de Homeodomínio , Lisina , Fatores de Transcrição , Acetilação , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lisina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Most human cancers harbor mutations in the gene encoding p53. As a result, research on p53 in the past few decades has focused primarily on its role as a tumor suppressor. One consequence of this focus is that the functions of p53 in development have largely been ignored. However, recent advances, such as the genomic profiling of embryonic stem cells, have uncovered the significance and mechanisms of p53 functions in mammalian cell differentiation and development. As we review here, these recent findings reveal roles that complement the well-established roles for p53 in tumor suppression.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Genes p53 , Proteína Supressora de Tumor p53/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Neoplasias/fisiopatologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Transcription factor p53 is activated in response to numerous stress stimuli in order to promote repair and survival or death of abnormal cells. For decades, regulatory mechanisms and downstream targets that execute the many biological functions of tumour suppressor p53 largely focused on the products of protein-coding genes. Recently, an entirely new class of molecules, termed long non-coding RNAs (lncRNAs), were discovered as key regulatory players in shaping p53 activity and biological outcomes. Many p53-regulated lncRNAs are now reported to either directly or indirectly intervene in p53-regulatory networks, generally in fine-tuning p53's tumour surveillance programme. Recent studies reveal that signals that converge upon p53 to regulate its activity, and molecules that implement downstream p53-response include both proteins and lncRNAs. In this review, we discuss the non-proteomic component of p53-regulatory networks, focusing on lncRNAs regulated by p53 and/or that regulate p53 activity, and their impact on biological outcomes.
Assuntos
Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Biomarcadores , Redes Reguladoras de Genes , Humanos , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
How tumor suppressor p53 selectively responds to specific signals, especially in normal cells, is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation, induced by retinoic acid, versus DNA damage, caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and, in pluripotent hESCs, are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases, UTX and JMJD3, to chromatin. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Dano ao DNA , Células-Tronco Embrionárias/citologia , Genoma Humano , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state.
Assuntos
Ciclo Celular , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Acetilação , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Apoptose , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , MicroRNAs/genética , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Transcrição Gênica , Tretinoína/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.
Assuntos
Diferenciação Celular , Ácidos Graxos , Miócitos Cardíacos , Miofibrilas , Fosforilação Oxidativa , Proteína 2 de Ligação ao Retinoblastoma , Humanos , Células Cultivadas , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Histonas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/enzimologia , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/genética , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Miofibrilas/enzimologia , Oxirredução , Regiões Promotoras Genéticas , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genéticaRESUMO
Rationale: Human pluripotent stem cell-derived CMs (iPSC-CMs) are a valuable tool for disease modeling, cell therapy and to reconstruct the CM maturation process and identify, characterize factors that regulate maturation. The transition from immature fetal to adult CM entails coordinated regulation of the mature gene programming, which is characterized by the induction of myofilament and OXPHOS gene expression among others. Recent studies in Drosophila , C. elegans, and C2C12 myoblast cell lines have implicated the histone H3K4me3 demethylase KDM5 and its homologs, as a potential regulator of developmental gene program and mitochondrial function. We speculated that KDM5 may potentiate the maturation of iPSC-CMs by targeting a conserved epigenetic program that encompass mitochondrial OXPHOS and other CM specific maturation genes. Objectives: The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. Methods and Results: Immunoblot analysis revealed that KDM5A, B, and C expression was progressively downregulated in postnatal cardiomyocytes and absent in adult hearts and CMs. Additionally, KDM5 proteins were found to be persistently expressed in iPSC-CMs up to 60 days after the onset of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor-resulted in differential regulation of 2,372 genes including upregulation of Fatty acid oxidation (FAO), OXPHOS, and myogenic gene programs in iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the CUT&RUN assay revealed enriched H3K4me3 peaks at the promoter regions of FAO, OXPHOS, and sarcomere genes. Consistent with the chromatin and gene expression data, KDM5 inhibition led to increased expression of multiple sarcomere proteins, enhanced myofibrillar organization and improved calcium handling. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene, which is known to regulate OXPHOS and cardiomyocyte maturation, and resulted in its increased RNA and protein levels. Finally, KDM5 inhibition increased baseline, peak, and spare oxygen consumption rates in iPSC-CMs. Conclusions: KDM5 regulates the maturation of iPSC-CMs by epigenetically regulating the expression of ESRRA, OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.
RESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of therapy resistance. Since the cell of origin can impact response to therapy, it is crucial to understand the lineage composition of AML cells at time of therapy resistance. Here we leverage single-cell chromatin accessibility profiling of 22 AML bone marrow aspirates from eight patients at time of therapy resistance and following subsequent therapy to characterize their lineage landscape. Our findings reveal a complex lineage architecture of therapy-resistant AML cells that are primed for stem and progenitor lineages and spanning quiescent, activated and late stem cell/progenitor states. Remarkably, therapy-resistant AML cells are also composed of cells primed for differentiated myeloid, erythroid and even lymphoid lineages. The heterogeneous lineage composition persists following subsequent therapy, with early progenitor-driven features marking unfavorable prognosis in The Cancer Genome Atlas AML cohort. Pseudotime analysis further confirms the vast degree of heterogeneity driven by the dynamic changes in chromatin accessibility. Our findings suggest that therapy-resistant AML cells are characterized not only by stem and progenitor states, but also by a continuum of differentiated cellular lineages. The heterogeneity in lineages likely contributes to their therapy resistance by harboring different degrees of lineage-specific susceptibilities to therapy.
Assuntos
Cromatina , Leucemia Mieloide Aguda , Humanos , Cromatina/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Diferenciação Celular , Divisão Celular , Linhagem da Célula/genéticaRESUMO
Oncogenic KRASG12D (KRAS∗) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC) and is a known repressor of tumor immunity. Conditional elimination of KRAS∗ in genetic mouse models of PDAC leads to the reactivation of FAS, CD8+ T cell-mediated apoptosis, and complete eradication of tumors. KRAS∗ elimination recruits activated CD4+ and CD8+ T cells and promotes the activation of antigen-presenting cells. Mechanistically, KRAS∗-mediated immune evasion involves the epigenetic regulation of Fas death receptor in cancer cells, via methylation of its promoter region. Furthermore, analysis of human RNA sequencing identifies that high KRAS expression in PDAC tumors shows a lower proportion of CD8+ T cells and demonstrates shorter survival compared with tumors with low KRAS expression. This study highlights the role of CD8+ T cells in the eradication of PDAC following KRAS∗ elimination and provides a rationale for the combination of KRAS∗ targeting with immunotherapy to control PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Animais , Humanos , Camundongos , Apoptose , Carcinoma Ductal Pancreático/genética , Linfócitos T CD8-Positivos , Epigênese Genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li-Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. SIGNIFICANCE: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li-Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are "basal" p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027.