RESUMEN
High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes SRCAP and P400-TIP60 in melanoma remains unclear. Here, we show that individual subunit depletion of SRCAP, P400, and VPS72 (YL1) results in not only the loss of H2A.Z deposition into chromatin but also a reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is particularly found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.
Asunto(s)
Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Histonas , Melanoma , Humanos , Melanoma/genética , Proliferación Celular/genética , Línea Celular Tumoral , Histonas/metabolismo , Histonas/genética , Acetilación , Apoptosis/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genéticaRESUMEN
Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a transmembrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.
Asunto(s)
Antineoplásicos/farmacología , Elementos de Facilitación Genéticos , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Histone variants are emerging as key regulatory molecules in cancer. We report a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses reveal that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We identify BRD2 as an H2A.Z-interacting protein, levels of which are also elevated in melanoma. We further demonstrate that H2A.Z.2-regulated genes are bound by BRD2 and E2F1 in an H2A.Z.2-dependent manner. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies.
Asunto(s)
Resistencia a Antineoplásicos/genética , Factor de Transcripción E2F1/genética , Histonas/genética , Melanoma/genética , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/genética , Factor de Transcripción E2F1/metabolismo , Células HeLa , Histonas/biosíntesis , Humanos , Melanocitos/citología , Melanoma/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Puntos de Control de la Fase S del Ciclo Celular/genética , Análisis de Secuencia de ARN , Factores de Transcripción , Activación TranscripcionalRESUMEN
Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Mitosis , Cresta Neural/embriología , Animales , Línea Celular , Humanos , Unión Proteica , Xenopus/embriologíaRESUMEN
The histone variant macroH2A generally associates with transcriptionally inert chromatin; however, the factors that regulate its chromatin incorporation remain elusive. Here, we identify the SWI/SNF helicase ATRX (α-thalassemia/MR, X-linked) as a novel macroH2A-interacting protein. Unlike its role in assisting H3.3 chromatin deposition, ATRX acts as a negative regulator of macroH2A's chromatin association. In human erythroleukemic cells deficient for ATRX, macroH2A accumulates at the HBA gene cluster on the subtelomere of chromosome 16, coinciding with the loss of α-globin expression. Collectively, our results implicate deregulation of macroH2A's distribution as a contributing factor to the α-thalassemia phenotype of ATRX syndrome.
Asunto(s)
Cromatina/metabolismo , ADN Helicasas/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Globinas alfa/genética , Globinas alfa/metabolismo , ADN Helicasas/genética , Células Eritroides/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Células K562 , Discapacidad Intelectual Ligada al Cromosoma X/patología , Proteínas Nucleares/genética , Telómero/metabolismo , Proteína Nuclear Ligada al Cromosoma X , Talasemia alfa/patologíaRESUMEN
Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Melanoma/patología , Metástasis de la Neoplasia/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HCT116 , Histonas/deficiencia , Histonas/genética , Humanos , Melanoma/fisiopatología , Melanoma Experimental , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Metástasis de la Neoplasia/fisiopatología , Ratas , Regulación hacia ArribaRESUMEN
Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology.
Asunto(s)
Cromatina/fisiología , Variación Genética , Chaperonas de Histonas/genética , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Neoplasias/genética , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Componentes del Gen , Chaperonas de Histonas/metabolismo , Histonas/química , Humanos , Neoplasias/fisiopatologíaRESUMEN
High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes, SRCAP and P400-TIP60, in melanoma remains unclear. Here, we show that individual depletion of SRCAP, P400, and VPS72 (YL1) not only results in loss of H2A.Z deposition into chromatin, but also a striking reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a highly coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.
RESUMEN
BACKGROUND: Despite the large amount of data available on the molecular mechanisms that regulate HIV-1 transcription, crucial information is still lacking about the interplay between chromatin conformation and the events that regulate initiation and elongation of viral transcription. During transcriptional activation, histone acetyltransferases and ATP-dependent chromatin remodeling complexes cooperate with histone chaperones in altering chromatin structure. In particular, human Nucleosome Assembly Protein-1 (hNAP-1) is known to act as a histone chaperone that shuttles histones H2A/H2B into the nucleus, assembles nucleosomes and promotes chromatin fluidity, thereby affecting transcription of several cellular genes. RESULTS: Using a proteomic screening, we identified hNAP-1 as a novel cellular protein interacting with HIV-1 Tat. We observed that Tat specifically binds hNAP1, but not other members of the same family of factors. Binding between the two proteins required the integrity of the basic domain of Tat and of two separable domains of hNAP-1 (aa 162-290 and 290-391). Overexpression of hNAP-1 significantly enhanced Tat-mediated activation of the LTR. Conversely, silencing of the protein decreased viral promoter activity. To explore the effects of hNAP-1 on viral infection, a reporter HIV-1 virus was used to infect cells in which hNAP-1 had been either overexpressed or knocked-down. Consistent with the gene expression results, these two treatments were found to increase and inhibit viral infection, respectively. Finally, we also observed that the overexpression of p300, a known co-activator of both Tat and hNAP-1, enhanced hNAP-1-mediated transcriptional activation as well as its interaction with Tat. CONCLUSION: Our study reveals that HIV-1 Tat binds the histone chaperone hNAP-1 both in vitro and in vivo and shows that this interaction participates in the regulation of Tat-mediated activation of viral gene expression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Regulación Viral de la Expresión Génica , Productos del Gen tat/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Línea Celular , Proteína p300 Asociada a E1A/metabolismo , Histonas , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteína 1 de Ensamblaje de Nucleosomas , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Secuencias Repetidas Terminales/genéticaRESUMEN
BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) favors integration in active genes of host chromatin. It is believed that transcriptional interference of the viral promoter over the endogenous gene or vice versa might occur with implications in HIV-1 post-integrative transcriptional latency. RESULTS: In this work a cell line has been transduced with a HIV-based vector and selected for Tat-inducible expression. These cells were found to carry a single silent integration in sense orientation within the second intron of the HMBOX1 gene. The HIV-1 Tat transactivator induced the viral LTR and repressed HMBOX1 expression independently of vector integration. Instead, single-cell quantitative in situ hybridization revealed that allele-specific transcription of HMBOX1 carrying the integrated provirus was not affected by the transactivation of the viral LTR in cis. CONCLUSION: A major observation of the work is that the HIV-1 genome has inserted in genes that are also repressed by Tat and this could be an advantage for the virus during transcriptional reactivation. In addition, it has also been observed that transcription of the provirus and of the endogenous gene in which it is integrated may coexist at the same time in the same genomic location.
Asunto(s)
VIH-1/fisiología , Proteínas de Homeodominio/biosíntesis , ARN Viral/biosíntesis , ARN/biosíntesis , Transcripción Genética , Integración Viral , Alelos , Línea Celular , Proteínas de Homeodominio/genética , Humanos , Hibridación Fluorescente in SituRESUMEN
Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Interleucinas/metabolismo , Melanoma/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Metilación de ADN , Sinergismo Farmacológico , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Melanoma/genética , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Mutación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Células THP-1 , Trasplante Heterólogo , Células U937RESUMEN
Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na+,K+-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.
Asunto(s)
Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Células CHO , Glicósidos Cardíacos/metabolismo , Cricetulus , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Histone variants are attracting attention in the field of cancer epigenetics. Our study has established a novel role for the uncharacterized histone variant H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 promotes cellular proliferation by recruiting BRD2 and E2F1 to E2F target genes and facilitating their transcription. High H2A.Z.2 expression correlates with poor survival in patients, and its depletion sensitizes cells to chemotherapy and targeted therapies.