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Am J Hum Genet ; 105(2): 283-301, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31353023


The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

Sci Rep ; 9(1): 8239, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160609


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.

Nat Struct Mol Biol ; 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30510221


TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5-TAF6-TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6-TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.

EMBO J ; 37(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29363506


The atypical E2Fs, E2F7 and E2F8, act as potent transcriptional repressors of DNA replication genes providing them with the ability to induce a permanent S-phase arrest and suppress tumorigenesis. Surprisingly in human cancer, transcript levels of atypical E2Fs are frequently elevated in proliferating cancer cells, suggesting that the tumor suppressor functions of atypical E2Fs might be inhibited through unknown post-translational mechanisms. Here, we show that atypical E2Fs can be directly phosphorylated by checkpoint kinase 1 (Chk1) to prevent a permanent cell cycle arrest. We found that 14-3-3 protein isoforms interact with both E2Fs in a Chk1-dependent manner. Strikingly, Chk1 phosphorylation and 14-3-3-binding did not relocate or degrade atypical E2Fs, but instead, 14-3-3 is recruited to E2F7/8 target gene promoters to possibly interfere with transcription. We observed that high levels of 14-3-3 strongly correlate with upregulated transcription of atypical E2F target genes in human cancer. Thus, we reveal that Chk1 and 14-3-3 proteins cooperate to inactivate the transcriptional repressor functions of atypical E2Fs. This mechanism might be of particular importance to cancer cells, since they are exposed frequently to DNA-damaging therapeutic reagents.

Proteínas 14-3-3/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Quinase 1 do Ponto de Checagem/metabolismo , Fator de Transcrição E2F7/antagonistas & inibidores , Neoplasias/patologia , Proteínas Repressoras/antagonistas & inibidores , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Replicação do DNA/genética , Fator de Transcrição E2F7/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Biossíntese de Proteínas/genética , Proteínas Repressoras/metabolismo
Nat Genet ; 49(11): 1642-1646, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28920961


Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

Reparo do DNA , Deficiências do Desenvolvimento/genética , Histonas/genética , Deficiência Intelectual/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Adolescente , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Criança , Dano ao DNA , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Instabilidade Genômica , Mutação em Linhagem Germinativa , Histonas/metabolismo , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Microcefalia/diagnóstico , Microcefalia/metabolismo , Microcefalia/patologia , Nucleossomos/química , Nucleossomos/metabolismo , Síndrome , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
Cell Rep ; 16(11): 3041-3051, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27626671


The balance between protein synthesis and protein breakdown is a major determinant of protein homeostasis, and loss of protein homeostasis is one of the hallmarks of aging. Here we describe pulsed SILAC-based experiments to estimate proteome-wide turnover rates of individual proteins. We applied this method to determine protein turnover rates in Caenorhabditis elegans models of longevity and Parkinson's disease, using both developing and adult animals. Whereas protein turnover in developing, long-lived daf-2(e1370) worms is about 30% slower than in controls, the opposite was observed in day 5 adult worms, in which protein turnover in the daf-2(e1370) mutant is twice as fast as in controls. In the Parkinson's model, protein turnover is reduced proportionally over the entire proteome, suggesting that the protein homeostasis network has a strong ability to adapt. The findings shed light on the relationship between protein turnover and healthy aging.

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Doença , Longevidade , Proteoma/metabolismo , Animais , Modelos Animais de Doenças , Ontologia Genética , Insulina/metabolismo , Marcação por Isótopo , Mutação/genética , Doença de Parkinson/patologia , Transdução de Sinais , Somatomedinas/metabolismo