RESUMEN
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
Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Chaperonas de Histonas/genética , Código de Histonas , Histonas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Cromatina/química , Cromatina/metabolismo , Proteínas Co-Represoras , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Repeticiones de Microsatélite , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Cultivo Primario de Células , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.
Asunto(s)
Empalme Alternativo , Histonas/genética , Histonas/fisiología , Nucleosomas/metabolismo , Adenosina Trifosfatasas/metabolismo , Línea Celular , Cromatina/metabolismo , ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Lisina Acetiltransferasa 5 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Histona Desacetilasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Proteínas Represoras/metabolismo , Acetilación , Animales , Proteínas Cromosómicas no Histona/genética , Células HEK293 , Histona Desacetilasas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Nucleosomas/metabolismo , ARN Interferente Pequeño , Proteínas Represoras/genética , TransactivadoresRESUMEN
Histone lysine (K) methylation has been shown to play a fundamental role in modulating chromatin architecture and regulation of gene expression. Here we report on the identification of histone H3K56, located at the pivotal, nucleosome DNA entry/exit point, as a novel methylation site that is evolutionary conserved. We identify trimethylation of H3K56 (H3K56me3) as a modification that is present during all cell cycle phases, with the exception of S-phase, where it is underrepresented on chromatin. H3K56me3 is a novel heterochromatin mark, since it is enriched at pericentromeres but not telomeres and is thereby similar, but not identical, to the localization of H3K9me3 and H4K20me3. Possibly due to H3 sequence similarities, Suv39h enzymes, responsible for trimethylation of H3K9, also affect methylation of H3K56. Similarly, we demonstrate that trimethylation of H3K56 is removed by members of the JMJD2 family of demethylases that also target H3K9me3. Furthermore, we identify and characterize mouse mJmjd2E and its human homolog hKDM4L as novel, functionally active enzymes that catalyze the removal of two methyl groups from trimethylated H3K9 and K56. H3K56me3 is also found in C. elegans, where it co-localizes with H3K9me3 in most, but not all, tissues. Taken together, our findings raise interesting questions regarding how methylation of H3K9 and H3K56 is regulated in different organisms and their functional roles in heterochromatin formation and/or maintenance.