RESUMEN
Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.
Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes , Proteínas de Ciclo Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1/genética , Exones , Humanos , Mutación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Factores de Transcripción/genéticaRESUMEN
Early Xenopus development is characterized by a poor correlation between global mRNA and protein abundances due to maternal mRNA and protein loading. Therefore, proteome profiling is necessary to study gene expression dynamics during early Xenopus development. In contrast to mammals, single Xenopus eggs and embryos contain enough protein to allow identification and quantification of thousands of proteins using mass spectrometry-based proteomics. In addition to investigating developmental processes, single egg or blastomere proteomes can be used to study cell-to-cell variability at an unprecedented depth. In this protocol, we describe a mass spectrometry-based proteomics approach for the identification and absolute quantification of Xenopus laevis egg or embryo proteomes, including sample preparation, peptide fractionation and separation, and data analysis.
Asunto(s)
Embrión no Mamífero/metabolismo , Espectrometría de Masas/métodos , Proteoma/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Fraccionamiento Químico , Análisis de Datos , Intercambio Iónico , Péptidos/metabolismo , Manejo de EspecímenesRESUMEN
Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfoma/metabolismo , Proteínas Nucleares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Linfoma/genética , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Dominios Proteicos , Estabilidad Proteica , Transporte de Proteínas , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , UbiquitinaciónRESUMEN
Ubiquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LC)-MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.
Asunto(s)
Biología Computacional/métodos , Ubiquitina/química , Proteínas Ubiquitinadas/análisis , Sitios de Unión , Proteínas Portadoras , Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Poliubiquitina , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteoma , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/química , Ubiquitinación/fisiologíaRESUMEN
Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Mitosis , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Cromátides/genética , Segregación Cromosómica , Proteínas de Unión al ADN , Fibroblastos/metabolismo , Células HEK293 , Chaperonas de Histonas/genética , Humanos , Ratones , Proteínas Oncogénicas/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Transfección , Quinasa Tipo Polo 1RESUMEN
Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling.
Asunto(s)
Espectrometría de Masas , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteómica/métodos , Transducción de Señal , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación , Animales , Sitios de Unión , Biología Computacional , Cisteína Endopeptidasas/metabolismo , Bases de Datos de Proteínas , Células Madre Embrionarias/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Ratones , Células-Madre Neurales/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ubiquitina Tiolesterasa , Neoplasias del Cuello Uterino/metabolismo , Flujo de TrabajoRESUMEN
Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-l-phenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments, and we identify protein-protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.
Asunto(s)
Azidas/química , Química Clic/métodos , Proteínas de Unión al ADN/aislamiento & purificación , Fenilalanina/análogos & derivados , Proteómica/métodos , Factor de Transcripción STAT1/aislamiento & purificación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Fenilalanina/química , Fenilalanina/genética , Factor de Transcripción STAT1/química , Factor de Transcripción STAT1/genéticaRESUMEN
During the past decades, mass spectrometry (MS)-based proteomics has become an important technology to identify protein-protein interactions (PPIs). The application of a quantitative filter in protein enrichments from crude lysates to discriminate bona fide interactors from background proteins has proved to be particularly powerful. Recently, many different approaches to identify PPIs have been developed, including proximity-ligation technology and global interactome profiling based on the co-behavior of protein complexes in biochemical purification or perturbation experiments. Furthermore, methodologies have been introduced that provide information regarding the stoichiometry and topology of detected PPIs. We review these novel methodologies and emphasize the need to miniaturize workflows to analyze protein interactions in biological and pathological contexts where sample amounts are limited.
Asunto(s)
Espectrometría de Masas , Mapeo de Interacción de Proteínas , Animales , Humanos , Ratones , Proteínas/análisis , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/metabolismo , ProteómicaRESUMEN
The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.
Asunto(s)
Metilación de ADN , Elementos de Facilitación Genéticos , Animales , Tipificación del Cuerpo , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Xenopus , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Unión al ADN/genética , Femenino , Técnicas de Inactivación de Genes , Células HeLa , Histonas/genética , Humanos , Complejos Multiproteicos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Represoras/genética , Proteína 2 de Unión a Retinoblastoma/genética , Proteína 2 de Unión a Retinoblastoma/metabolismo , Factores de Transcripción/genéticaRESUMEN
Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Although histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells. The sNASP and ASF1 chaperones play pivotal roles in the processing of soluble histones but do not associate with the active CDC45/MCM2-7/GINS (CMG) replicative helicase. We also find TONSL-MMS22L to function as a H3-H4 histone chaperone. It associates with the regulatory MCM5 subunit of the replicative helicase.
Asunto(s)
Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Espectrometría de Masas/métodos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Proteínas de Mantenimiento de Minicromosoma/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Unión ProteicaRESUMEN
While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Proteoma/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Desarrollo Embrionario/genética , Óvulo/metabolismo , Proteoma/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual , Transcriptoma , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.
Asunto(s)
Histona Desacetilasas/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Proteínas Represoras/química , Proteína 4 de Unión a Retinoblastoma/química , Secuencia de Aminoácidos , Animales , Ensamble y Desensamble de Cromatina , Secuencia Conservada , Cristalografía por Rayos X , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Proteína 7 de Unión a Retinoblastoma/química , Proteína 7 de Unión a Retinoblastoma/genética , Proteína 7 de Unión a Retinoblastoma/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
MBD5 and MBD6 are two members of the methyl-CpG-binding domain (MBD) family of proteins that are poorly characterized. Studies performed thus far have failed to show binding of the MBD5 and MBD6 MBD to methylated DNA. Here, we show that both MBD5 and MBD6 interact with the mammalian PR-DUB Polycomb protein complex in a mutually exclusive manner. Strikingly, the MBD of MBD5 and MBD6 is both necessary and sufficient to mediate this interaction. Chromatin immunoprecipitation analyses reveal that MBD6 and FOXK2/PR-DUB share a subset of genomic target genes, suggesting a functional interaction in vivo. Finally, we show that MBD6, but not MBD5, is recruited to sites of DNA damage in a PR-DUB independent manner. Our study thus implies a shared function for MBD5 and MBD6 through an interaction with PR-DUB, as well as an MBD6-specific recruitment to sites of DNA damage.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Secuencia de Aminoácidos , Cromatina , Daño del ADN , Metilación de ADN , Factores de Transcripción Forkhead , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
X chromosome inactivation is a remarkable example of chromosome-wide gene silencing and facultative heterochromatin formation. Numerous histone posttranslational modifications, including H3K9me2 and H3K27me3, accompany this process, although our understanding of the enzymes that lay down these marks and the factors that bind to them is still incomplete. Here we identify Cdyl, a chromodomain-containing transcriptional corepressor, as a new chromatin-associated protein partner of the inactive X chromosome (Xi). Using mouse embryonic stem cell lines with mutated histone methyltransferase activities, we show that Cdyl relies on H3K9me2 for its general association with chromatin in vivo. For its association with Xi, Cdyl requires the process of differentiation and the presence of H3K9me2 and H3K27me3, which both become chromosomally enriched following Xist RNA coating. We further show that the removal of the PRC2 component Eed and subsequent loss of H3K27me3 lead to a reduction of both Cdyl and H3K9me2 enrichment on inactive Xi. Finally, we show that Cdyl associates with the H3K9 histone methyltransferase G9a and the MGA protein, both of which are also found on Xi. We propose that the combination of H3K9me2 and H3K27me3 recruits Cdyl to Xi, and this, in turn, may facilitate propagation of the H3K9me2 mark by anchoring G9a.
Asunto(s)
Células Madre Embrionarias/fisiología , Histonas/metabolismo , Proteínas/metabolismo , Inactivación del Cromosoma X , Cromosoma X/metabolismo , Animales , Diferenciación Celular , Núcleo Celular/metabolismo , Proteínas Co-Represoras , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Histona Acetiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Hidroliasas , Metilación , Ratones , Complejo Represivo Polycomb 1/metabolismo , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Tretinoina/fisiología , Cromosoma X/genéticaRESUMEN
Methylation of lysine 4 on histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multisubunit (SET1/MLL) complexes that contain methyltransferase activity for H3K4 have been described, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. We identified both known and novel, unique and shared interactors and determined their distribution and stoichiometry over the different SET1/MLL complexes. In addition to being a core COMPASS subunit, the Dpy30 protein is a genuine subunit of the NURF chromatin remodeling complex. Furthermore, we identified the Bod1 protein as a discriminator between the SET1B and SET1A complexes, and we show that the H3K36me-interactor Psip1 preferentially binds to the MLL2 complex. Finally, absolute protein quantification in crude lysates mirrors many of the observed SET1/MLL complex stoichiometries. Our findings provide a molecular framework for understanding the diversity and abundance of the different SET1/MLL complexes, which together establish the H3K4 methylation landscape in human cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Neoplasias/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatografía de Afinidad , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , N-Metiltransferasa de Histona-Lisina/aislamiento & purificación , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína de la Leucemia Mieloide-Linfoide/aislamiento & purificación , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Neoplasias/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Mapeo de Interacción de Proteínas , Subunidades de Proteína/aislamiento & purificación , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismoRESUMEN
Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.
Asunto(s)
5-Metilcitosina/análisis , Citosina/análogos & derivados , Metilación de ADN , 5-Metilcitosina/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Citosina/análisis , Citosina/metabolismo , ADN Glicosilasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Factor 4 Similar a Kruppel , Espectrometría de Masas , Ratones , Oxidación-Reducción , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción del Factor Regulador X , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Histone variants are key components of the epigenetic code and evolved to perform specific functions in transcriptional regulation, DNA repair, chromosome segregation and other fundamental processes. Although variants for histone H2A and H3 are found throughout the eukaryotic kingdom, variants of histone H2B and H4 are rarely encountered. H2B.Z is one of those rare H2B variants and is apicomplexan-specific. Here we show that in Plasmodium falciparum H2B.Z localizes to euchromatic intergenic regions throughout intraerythrocytic development and together with H2A.Z forms a double-variant nucleosome subtype. These nucleosomes are enriched in promoters over 3' intergenic regions and their occupancy generally correlates with the strength of the promoter, but not with its temporal activity. Remarkably, H2B.Z occupancy levels exhibit a clear correlation with the base-composition of the underlying DNA, raising the intriguing possibility that the extreme AT content of the intergenic regions within the Plasmodium genome might be instructive for histone variant deposition. In summary, our data show that the H2A.Z/H2B.Z double-variant nucleosome demarcates putative regulatory regions of the P. falciparum epigenome and likely provides a scaffold for dynamic regulation of gene expression in this deadly human pathogen.
Asunto(s)
Secuencia Rica en At , Genoma de Protozoos , Histonas/metabolismo , Nucleosomas/genética , Plasmodium falciparum/genética , Regiones Promotoras Genéticas , Proteínas Protozoarias/metabolismo , Secuencia de Bases , ADN Intergénico , Histonas/genética , Humanos , Malaria Falciparum/parasitología , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Plasmodium falciparum/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genéticaRESUMEN
Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.
Asunto(s)
Cromatina/química , Proteínas Cromosómicas no Histona/análisis , Espectrometría de Masas , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Proteínas de Ciclo Celular/análisis , Enzimas Reparadoras del ADN/análisis , Proteínas de Unión al ADN/análisis , Células HeLa , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/análisis , Proteínas Nucleares/análisis , Complejo Represivo Polycomb 2/análisis , Subunidades de Proteína/análisis , Factores de Empalme de ARN , Proteínas de Unión al ARN/análisisRESUMEN
Centromeres are essential for the faithful transmission of chromosomes to the next generation, therefore being essential in all eukaryotic organisms. The centromeres of Plasmodium falciparum, the causative agent of the most severe form of malaria, have been broadly mapped on most chromosomes, but their epigenetic composition remained undefined. Here, we reveal that the centromeric histone variant PfCENH3 occupies a 4-4.5 kb region on each P. falciparum chromosome, which is devoid of pericentric heterochromatin but harbours another histone variant, PfH2A.Z. These CENH3 covered regions pinpoint the exact position of the centromere on all chromosomes and revealed that all centromeric regions have similar size and sequence composition. Immunofluorescence assay of PfCENH3 strongly suggests that P. falciparum centromeres cluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion. In summary, we reveal a dynamic association of Plasmodium centromeres, which bear a unique epigenetic signature and conform to a strict structure. These findings suggest that DNA-associated and epigenetic elements play an important role in centromere establishment in this important human pathogen.