RESUMO
Proximity-dependent biotin labeling (BioID) may identify new targets for cancers driven by difficult-to-drug oncogenes such as Ras. Therefore, BioID was used with wild-type (WT) and oncogenic mutant (MT) H-, K-, and N-Ras, identifying known interactors, including Raf and PI3K, as well as a common set of 130 novel proteins proximal to all Ras isoforms. A CRISPR screen of these proteins for Ras dependence identified mTOR, which was also found proximal to MT Ras in human tumors. Oncogenic Ras directly bound two mTOR complex 2 (mTORC2) components, mTOR and MAPKAP1, to promote mTORC2 kinase activity at the plasma membrane. mTORC2 enabled the Ras pro-proliferative cell cycle transcriptional program, and perturbing the Ras-mTORC2 interaction impaired Ras-dependent neoplasia in vivo. Combining proximity-dependent proteomics with CRISPR screening identified a new set of functional Ras-associated proteins, defined mTORC2 as a new direct Ras effector, and offers a strategy for finding new proteins that cooperate with dominant oncogenes.
Assuntos
Transformação Celular Neoplásica/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Neoplasias/metabolismo , Proteoma , Proteínas ras/metabolismo , Animais , Sítios de Ligação , Sistemas CRISPR-Cas , Células CACO-2 , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos Pelados , Camundongos SCID , Camundongos Transgênicos , Mutação , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteômica/métodos , Carga Tumoral , Proteínas ras/genéticaRESUMO
The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14.
Assuntos
Reparo do DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Histonas/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIID/metabolismo , Acetilação , Dano ao DNA , Histonas/química , Histonas/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
Monoubiquitylation of histone H2B on Lys123 (H2BK123ub1) plays a multifaceted role in diverse DNA-templated processes, yet the mechanistic details by which this modification is regulated are not fully elucidated. Here we show in yeast that H2BK123ub1 is regulated in part through the protein stability of the E3 ubiquitin ligase Bre1. We found that Bre1 stability is controlled by the Rtf1 subunit of the polymerase-associated factor (PAF) complex and through the ability of Bre1 to catalyze H2BK123ub1. Using a domain in Rtf1 that stabilizes Bre1, we show that inappropriate Bre1 levels lead to defects in gene regulation. Collectively, these data uncover a novel quality control mechanism used by the cell to maintain proper Bre1 and H2BK123ub1 levels, thereby ensuring proper control of gene expression.
Assuntos
Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Catálise , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/metabolismo , UbiquitinaçãoRESUMO
Bypass of Ess1 (Bye1) is a nuclear protein with a domain resembling the central domain in the transcription elongation factor TFIIS. Here we show that Bye1 binds with its TFIIS-like domain (TLD) to RNA polymerase (Pol) II, and report crystal structures of the Bye1 TLD bound to Pol II and three different Pol II-nucleic acid complexes. Like TFIIS, Bye1 binds with its TLD to the Pol II jaw and funnel. In contrast to TFIIS, however, it neither alters the conformation nor the in vitro functions of Pol II. In vivo, Bye1 is recruited to chromatin via its TLD and occupies the 5'-region of active genes. A plant homeo domain (PHD) in Bye1 binds histone H3 tails with trimethylated lysine 4, and this interaction is enhanced by the presence of neighboring posttranslational modifications (PTMs) that mark active transcription and conversely is impaired by repressive PTMs. We identify putative human homologs of Bye1, the proteins PHD finger protein 3 and death-inducer obliterator, which are both implicated in cancer. These results establish Bye1 as the founding member of a unique family of chromatin transcription factors that link histones with active PTMs to transcribing Pol II.
Assuntos
Cromatina/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , RNA Polimerase II/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica/fisiologia , Fatores de Elongação da Transcrição/química , Escherichia coli , Análise em Microsséries , Complexos Multiproteicos/metabolismo , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Conformação Proteica , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de Elongação da Transcrição/metabolismoRESUMO
The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification.
Assuntos
Alquil e Aril Transferases/metabolismo , Inativação Gênica/fisiologia , RNA Polimerase II/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alquil e Aril Transferases/genética , Arabidopsis , Atorvastatina , Northern Blotting , Nucléolo Celular/metabolismo , Imunoprecipitação da Cromatina , Clonagem Molecular , Primers do DNA/genética , Ácidos Heptanoicos , Humanos , Imunoprecipitação , Hibridização In Situ , Oligonucleotídeos/genética , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Pirróis , RNA Polimerase II/fisiologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Histones and their posttranslational modifications (PTMs) play an important role in regulating DNA-templated processes. While some PTMs directly modulate chromatin architecture via charge effects, others rely on the action of reader or effector proteins that can recognize and bind the modification to fulfill distinct cellular outcomes. One PTM that has been well studied with regard to reader proteins is histone lysine methylation - a PTM linked to many DNA-templated processes including transcription, DNA replication and DNA repair. In this review, we summarize the current understanding of how histone lysine methylation is read during the process of active transcription. We also describe how the interpretation of lysine methylation fits into a larger, more complex 'code' of histone PTMs to modulate chromatin structure and function. These insights take into account emerging concepts in the field in an effort to help facilitate future studies.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Animais , Metilação de DNA , Histona Metiltransferases , Humanos , Metilação , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Widespread changes in gene expression underlie B cell development and activation, yet our knowledge of which chromatin-remodeling factors are essential is limited. Here, we demonstrate that the BRG1 catalytic subunit of SWI/SNF complexes was dispensable for murine B cell development but played an important, albeit selective, role during activation. Although BRG1 was dispensable for CD69 induction and differentiation into plasma cells based on the ability of mutant B cells to undergo hypertrophy and secrete IgM antibodies, it was required for robust cell proliferation in response to activation. Accordingly, BRG1 was required for only â¼100 genes to be expressed at normal levels in naïve B cells but >1,000 genes during their activation. BRG1 upregulated fivefold more genes than it downregulated, and the toll-like receptor pathway and JAK/STAT cytokine-signaling pathways were particularly dependent on BRG1. The importance of BRG1 in B cell activation was underscored by the occurrence of opportunistic Pasteurella infections in conditionally mutant mice. B cell activation has long served as a model of inducible gene expression, and the results presented here identify BRG1 as a chromatin-remodeling factor that upregulates the transcriptome of B cells during their activation to promote rapid cell proliferation and to mount an effective immune response.
Assuntos
Linfócitos B/metabolismo , Montagem e Desmontagem da Cromatina/genética , DNA Helicases , Ativação Linfocitária/genética , Proteínas Nucleares , Fatores de Transcrição , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/fisiologia , Diferenciação Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Loss of function of progranulin (PGRN), encoded by the granulin (GRN) gene, is implicated in several neurodegenerative diseases. Several therapeutics to boost PGRN levels are currently in clinical trials. However, it is difficult to test the efficacy of PGRN-enhancing drugs in mouse models due to the mild phenotypes of Grn-/- mice. Recently, mice deficient in both PGRN and TMEM106B were shown to develop severe motor deficits and pathology. Here, we show that intracerebral ventricle injection of PGRN-expressing AAV1/9 viruses partially rescues motor deficits, neuronal loss, glial activation, and lysosomal abnormalities in Tmem106b-/-Grn-/- mice. Widespread expression of PGRN is detected in both the brain and spinal cord for both AAV subtypes. However, AAV9 but not AAV1-mediated expression of PGRN results in high levels of PGRN in the serum. Together, these data support using the Tmem106b-/-Grn-/- mouse strain as a robust mouse model to determine the efficacy of PGRN-elevating therapeutics.
RESUMO
KRAS receives and relays signals at the plasma membrane (PM) where it transmits extracellular growth factor signals to downstream effectors. SNORD50A/B were recently found to bind KRAS and inhibit its tumorigenic action by unknown mechanisms. KRAS proximity protein labeling was therefore undertaken in SNORD50A/B wild-type and knockout cells, revealing that SNORD50A/B RNAs shape the composition of proteins proximal to KRAS, notably by inhibiting KRAS proximity to the SNARE vesicular transport proteins SNAP23, SNAP29, and VAMP3. To remain enriched on the PM, KRAS undergoes cycles of endocytosis, solubilization, and vesicular transport to the PM. Here we report that SNAREs are essential for the final step of this process, with KRAS localization to the PM facilitated by SNAREs but antagonized by SNORD50A/B. Antagonism between SNORD50A/B RNAs and specific SNARE proteins thus controls KRAS localization, signaling, and tumorigenesis, and disrupting SNARE-enabled KRAS function represents a potential therapeutic opportunity in KRAS-driven cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas p21(ras)/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas SNARE/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pequeno RNA não Traduzido/genética , Transdução de SinaisRESUMO
Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.