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Polycomb repressive complex 2 (PRC2) mediates H3K27me3 deposition, which is thought to recruit canonical PRC1 (cPRC1) via chromodomain-containing CBX proteins to promote stable repression of developmental genes. PRC2 forms two major subcomplexes, PRC2.1 and PRC2.2, but their specific roles remain unclear. Through genetic knockout (KO) and replacement of PRC2 subcomplex-specific subunits in naïve and primed pluripotent cells, we uncover distinct roles for PRC2.1 and PRC2.2 in mediating the recruitment of different forms of cPRC1. PRC2.1 catalyzes the majority of H3K27me3 at Polycomb target genes and is sufficient to promote recruitment of CBX2/4-cPRC1 but not CBX7-cPRC1. Conversely, while PRC2.2 is poor at catalyzing H3K27me3, we find that its accessory protein JARID2 is essential for recruitment of CBX7-cPRC1 and the consequent 3D chromatin interactions at Polycomb target genes. We therefore define distinct contributions of PRC2.1- and PRC2.2-specific accessory proteins to Polycomb-mediated repression and uncover a new mechanism for cPRC1 recruitment.
Asunto(s)
Histonas , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Histonas/genética , Histonas/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Cromatina/genéticaRESUMEN
DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.
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Ensamble y Desensamble de Cromatina , Cromatina , Proteínas Nucleares , Nucleosomas , Proteómica , Humanos , Sitios de Unión , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteómica/métodosRESUMEN
ADP-ribose (ADPr) readers are essential components of ADP-ribosylation signaling, which regulates genome maintenance and immunity. The identification and discrimination between monoADPr (MAR) and polyADPr (PAR) readers is difficult because of a lack of suitable affinity-enrichment reagents. We synthesized well-defined ADPr probes and used these for affinity purifications combined with relative and absolute quantitative mass spectrometry to generate proteome-wide MAR and PAR interactomes, including determination of apparent binding affinities. Among the main findings, MAR and PAR readers regulate various common and distinct processes, such as the DNA-damage response, cellular metabolism, RNA trafficking, and transcription. We monitored the dynamics of PAR interactions upon induction of oxidative DNA damage and uncovered the mechanistic connections between ubiquitin signaling and ADP-ribosylation. Taken together, chemical biology enables exploration of MAR and PAR readers using interaction proteomics. Furthermore, the generated MAR and PAR interaction maps significantly expand our current understanding of ADPr signaling.
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ADP-Ribosilación , Adenosina Difosfato Ribosa/química , Adenosina Difosfato/química , Proteómica/métodos , Ubiquitina-Proteína Ligasas/química , Sitio Alostérico , Animales , Anticuerpos Monoclonales/química , Sitios de Unión , Biotinilación , Comunicación Celular , Daño del ADN , Técnicas Genéticas , Células HeLa , Humanos , Espectrometría de Masas , Ratones , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteoma , Transducción de Señal , UbiquitinaRESUMEN
Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.
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Composición de Base , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Ratones , Ratones Mutantes , Células Madre Embrionarias de Ratones/citología , Mutación , Neuronas/citología , Factores de Transcripción/genética , Regulación hacia Arriba , Dedos de ZincRESUMEN
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.
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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
Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.
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Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/enzimología , Mutación , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Biosíntesis de Proteínas/genética , ARN Ribosómico 18S/metabolismoRESUMEN
Estrogen Receptor 1 (ESR1; also known as ERα, encoded by ESR1 gene) is the main driver and prime drug target in luminal breast cancer. ESR1 chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ESR1 chromatin action, along with its biological implications. Here, we use a large set of ESR1 ChIP-seq data from 70 ESR1+ breast cancers to explore inter-patient heterogeneity in ESR1 DNA binding to reveal a striking inter-tumor heterogeneity of ESR1 action. Of note, commonly shared ESR1 sites show the highest estrogen-driven enhancer activity and are most engaged in long-range chromatin interactions. In addition, the most commonly shared ESR1-occupied enhancers are enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ESR1 and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we can confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ESR1-chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ESR1 landscape, with the most common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.
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Neoplasias de la Mama , Cromatina , Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Cromatina/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Polimorfismo de Nucleótido SimpleRESUMEN
The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.
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Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Islas de CpG/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Cromatina/química , Ensamble y Desensamble de Cromatina/genética , Metilación de ADN , Regulación de la Expresión Génica , Genes Esenciales , Humanos , Ratones , Imagen Individual de MoléculaRESUMEN
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
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Codón sin Sentido/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , Codón sin Sentido/química , Exones/genética , Humanos , Intrones/genética , Mutación/genética , Estabilidad del ARN/genética , ARN Mensajero/química , Imagen Individual de MoléculaRESUMEN
Polycomb repressive complex 2 (PRC2) is composed of EED, SUZ12, and EZH1/2 and mediates mono-, di-, and trimethylation of histone H3 at lysine 27. At least two independent subcomplexes exist, defined by their specific accessory proteins: PRC2.1 (PCL1-3, EPOP, and PALI1/2) and PRC2.2 (AEBP2 and JARID2). We show that PRC2.1 and PRC2.2 share the majority of target genes in mouse embryonic stem cells. The loss of PCL1-3 is sufficient to evict PRC2.1 from Polycomb target genes but only leads to a partial reduction of PRC2.2 and H3K27me3. Conversely, disruption of PRC2.2 function through the loss of either JARID2 or RING1A/B is insufficient to completely disrupt targeting of SUZ12 by PCLs. Instead, the combined loss of both PRC2.1 and PRC2.2 is required, leading to the global mislocalization of SUZ12. This supports a model in which the specific accessory proteins within PRC2.1 and PRC2.2 cooperate to direct H3K27me3 via both synergistic and independent mechanisms.
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Cromatina/metabolismo , Histonas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Sitios de Unión , Línea Celular Tumoral , Cromatina/genética , Humanos , Metilación , Ratones , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/genética , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
DNA methylation has long been considered the primary epigenetic mediator of genomic imprinting in mammals. Recent epigenetic profiling during early mouse development revealed the presence of domains of trimethylation of lysine 27 on histone H3 (H3K27me3) and chromatin compaction specifically at the maternally derived allele, independent of DNA methylation. Within these domains, genes are exclusively expressed from the paternally derived allele. This novel mechanism of noncanonical imprinting plays a key role in the development of mouse extraembryonic tissues and in the regulation of imprinted X-chromosome inactivation, highlighting the importance of parentally inherited epigenetic histone modifications. Here, we discuss the mechanisms underlying H3K27me3-mediated noncanonical imprinting in perspective of the dynamic chromatin landscape during early mouse development and explore evolutionary origins of noncanonical imprinting.
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Impresión Genómica , Histonas , Animales , Cromatina/genética , Metilación de ADN/genética , Impresión Genómica/genética , Código de Histonas , Histonas/genética , Histonas/metabolismo , RatonesRESUMEN
Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in Ë 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.
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Inestabilidad Cromosómica/genética , Cromosomas/genética , Mutación/genética , Empalmosomas/genética , Secuencia de Aminoácidos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Células HeLa , Humanos , Intrones/genéticaRESUMEN
N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.
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Adenosina/análogos & derivados , Axones/fisiología , Proteínas de Drosophila/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Neuronas/citología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Neuronas/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify "crosstalk" between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding "profile" for proteins regulated by DNA and histone methylation.
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Ensamble y Desensamble de Cromatina , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Línea Celular Tumoral , Islas de CpG , Proteínas F-Box/metabolismo , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Metilación , Nucleosomas/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Proteómica/métodos , Coloración y Etiquetado/métodosRESUMEN
Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned to complexes by interaction proteomics of full-length BAC-GFP-tagged proteins. ChIP-Seq profiling identifies their genomic binding sites, revealing functional properties. Among the main findings, the human SAGA complex binds to H3K4me3 via a double Tudor-domain in the C terminus of Sgf29, and the PWWP domain is identified as a putative H3K36me3 binding motif. The ORC complex, including LRWD1, binds to the three most prominent transcriptional repressive lysine methylation sites. Our data reveal a highly adapted interplay between chromatin marks and their associated protein complexes. Reading specific trimethyl-lysine sites by specialized complexes appears to be a widespread mechanism to mediate gene expression.
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Cromatina/metabolismo , Epigénesis Genética , Código de Histonas , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células HeLa , Histona Acetiltransferasas/metabolismo , Humanos , Lisina/metabolismo , Espectrometría de Masas , Metilación , Proteómica/métodosRESUMEN
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.
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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.