RESUMO
Mutations in the RAS genes are identified in a variety of clinical settings, ranging from somatic mutations in oncology to germline mutations in developmental disorders, also known as 'RASopathies', and vascular malformations/overgrowth syndromes. Generally single amino acid substitutions are identified, that result in an increase of the GTP bound fraction of the RAS proteins causing constitutive signalling. Here, a series of 7 in-frame insertions and duplications in HRAS (n = 5) and KRAS (n = 2) is presented, resulting in the insertion of 7-10 amino acids residues in the switch II region. These variants were identified in routine diagnostic screening of 299 samples for somatic mutations in vascular malformations/overgrowth syndromes (n = 6) and in germline analyses for RASopathies (n = 1). Biophysical characterization shows the inability of Guanine Nucleotide Exchange Factors to induce GTP loading and reduced intrinsic and GAP-stimulated GTP hydrolysis. As a consequence of these opposing effects, increased RAS signalling is detected in a cellular model system. Therefore these in-frame insertions represent a new class of weakly activating clinically relevant RAS variants.
Assuntos
Mutação da Fase de Leitura/genética , Mutagênese Insercional/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Sequência de Aminoácidos , Estudos de Coortes , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Proteínas Mutantes/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/químicaRESUMO
Histone methylation patterns are correlated with eukaryotic gene transcription. High-affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine-4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high-affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3-PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3-mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live-cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho-methyl switch regulates TFIID-mediated transcription during mitotic progression of the cell cycle.
Assuntos
Histonas/genética , Mitose , Fator de Transcrição TFIID/genética , Ativação Transcricional , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromossomos/genética , Cromossomos/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Fator de Transcrição TFIID/metabolismoRESUMO
Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-pi interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 "methyl-methyl" switch in the histone code dynamically regulates TFIID-promoter association.
Assuntos
Histonas/química , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Animais , Análise Mutacional de DNA , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Metilação , Camundongos , Modelos Moleculares , Complexos Multiproteicos/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismoRESUMO
Trimethylation of histone H3 at lysine 4 (H3K4me3) is regarded as a hallmark of active human promoters, but it remains unclear how this posttranslational modification links to transcriptional activation. Using a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic screening we show that the basal transcription factor TFIID directly binds to the H3K4me3 mark via the plant homeodomain (PHD) finger of TAF3. Selective loss of H3K4me3 reduces transcription from and TFIID binding to a subset of promoters in vivo. Equilibrium binding assays and competition experiments show that the TAF3 PHD finger is highly selective for H3K4me3. In transient assays, TAF3 can act as a transcriptional coactivator in a PHD finger-dependent manner. Interestingly, asymmetric dimethylation of H3R2 selectively inhibits TFIID binding to H3K4me3, whereas acetylation of H3K9 and H3K14 potentiates TFIID interaction. Our experiments reveal crosstalk between histone modifications and the transcription factor TFIID. This has important implications for regulation of RNA polymerase II-mediated transcription in higher eukaryotes.