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1.
Neurogenetics ; 20(3): 129-143, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31041561

RESUMO

We previously reported a pathogenic de novo p.R342W mutation in the transcriptional corepressor CTBP1 in four independent patients with neurodevelopmental disabilities [1]. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CTBP1 mutation. Within this cohort, we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia, and tooth enamel defects present in most patients. The R342W mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin-modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cell lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.

2.
Front Immunol ; 10: 479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30936877

RESUMO

Background: HOIP is the catalytic subunit of the linear ubiquitination chain assembly complex (LUBAC) that is essential for NF-κB signaling and thus proper innate and adaptive immunity. To date only one patient with HOIP deficiency has been reported with clinical characteristics that include autoinflammation, immunodeficiency, amylopectinosis, and systemic lymphangiectasia. Case: We sought to identify a genetic cause of a disease for an 8 year-old girl who presented with early-onset immune deficiency and autoinflammation. Methods: Targeted next generation sequencing of 352 immune-related genes was performed. Functional studies included transcriptome analysis, cytokine profiling, and protein analysis in patients' primary cells. Results: We identified biallelic variants in close proximity to splice sites (c.1197G>C and c.1737+3A>G) in the RNF31 gene. RNA extracted from patient cells showed alternatively spliced transcripts not present in control cells. Protein expression of HOIP and LUBAC was reduced in primary cells as shown by western blotting. Patient-derived fibroblasts demonstrated attenuated IL-6 production, while PBMCs showed higher TNF production after stimulation with proinflammatory cytokines. RNA sequencing of whole blood RNA and PBMCs demonstrated a marked transcriptome wide change including differential expression of type I interferon regulated genes. Conclusion: We report the second case of HOIP deficiency with novel compound heterozygous mutations in RNF31 and distinct clinical and molecular features. Our results expand on the clinical spectrum of HOIP deficiency and molecular signatures associated with LUBAC deficiency.

3.
Front Immunol ; 10: 101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30766537

RESUMO

Monogenic autoinflammatory disorders are a group of conditions defined by systemic or localized inflammation without identifiable causes, such as infection. In contrast to classical primary immunodeficiencies that manifest with impaired immune responses, these disorders are due to defects in genes that regulate innate immunity leading to constitutive activation of pro-inflammatory signaling. Through studying patients with rare autoinflammatory conditions, novel mechanisms of inflammation have been identified that bare on our understanding not only of basic signaling in inflammatory cells, but also of the pathogenesis of more common inflammatory diseases and have guided treatment modalities. Autoinflammation has further been implicated as an important component of cardiovascular, neurodegenerative, and metabolic syndromes. In this review, we will focus on a subset of inherited enzymatic deficiencies that lead to constitutive inflammation, and how these rare diseases have provided insights into diverse areas of cell biology not restricted to immune cells. In this way, Mendelian disorders of the innate immune system, and in particular loss of catalytic activity of enzymes in distinct pathways, have expanded our understanding of the interplay between many seemingly disparate cellular processes. We also explore the overlap between autoinflammation, autoimmunity, and immunodeficiency, which has been increasingly recognized in patients with dysregulated immune responses.

4.
Am J Hum Genet ; 103(1): 100-114, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29979980

RESUMO

The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.

5.
Transl Sci Rare Dis ; 3(1): 45-48, 2018 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-29682452

RESUMO

Sengers syndrome is a rare autosomal recessive mitochondrial disease characterized by lactic acidosis, hypertrophic cardiomyopathy and bilateral cataracts. We present here a case of neonatal demise, within the first day of life, who initially presented with severe lactic acidosis, with evidence of both chorioamnionitis and cardiogenic shock. Initial metabolic labs demonstrated a severe lactic acidosis prompting genetic testing which revealed a homozygous pathogenic variant for Sengers syndrome in AGK, c.979A >  T; p.K327*. In addition to the canonical features of Sengers syndrome, our patient is the first reported case with liver dysfunction extending the phenotypic spectrum both in terms of severity and complications. This case also highlights the importance of maintaining a broad differential for congenital lactic acidosis.

6.
Curr Protoc Chem Biol ; 9(2): 128-146, 2017 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-28628203

RESUMO

Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest. In IPL, a heterobifunctional probe comprising a photoactivatable moiety and biotin is recruited by a monomeric streptavidin tag fused to a protein of interest. Following UV irradiation, candidate interacting proteins and RNAs are covalently biotinylated with tight spatial and temporal control and subsequently recovered using biotin as an affinity handle. Here, we describe experimental protocols to discover novel protein-protein and protein-RNA interactions using IPL. © 2017 by John Wiley & Sons, Inc.


Assuntos
Luz , Proteínas/metabolismo , Coloração e Rotulagem/métodos , Animais , Biotinilação , Células CHO , Cricetinae , Cricetulus , DNA/metabolismo , Células HEK293 , Humanos , Células Jurkat , RNA/metabolismo , Estreptavidina/metabolismo
7.
Neurogenetics ; 17(3): 173-8, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27094857

RESUMO

Exome sequencing is an effective way to identify genetic causes of etiologically heterogeneous conditions such as developmental delay and intellectual disabilities. Using exome sequencing, we have identified four patients with similar phenotypes of developmental delay, intellectual disability, failure to thrive, hypotonia, ataxia, and tooth enamel defects who all have the same de novo R331W missense variant in C-terminal binding protein 1 (CTBP1). CTBP1 is a transcriptional regulator critical for development by coordinating different regulatory pathways. The R331W variant found in these patients is within the C-terminal portion of the PLDLS (Pro-Leu-Asp-Leu-Ser) binding cleft, which is the domain through which CTBP1, interacts with chromatin-modifying enzymes and mediates chromatin-dependent gene repression pathways. This is the first report of mutations within CTBP1 in association with any human disease.


Assuntos
Oxirredutases do Álcool/genética , Ataxia/genética , Proteínas de Ligação a DNA/genética , Esmalte Dentário/patologia , Deficiências do Desenvolvimento/genética , Hipotonia Muscular/genética , Mutação de Sentido Incorreto , Adulto , Ataxia/complicações , Criança , Deficiências do Desenvolvimento/complicações , Feminino , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Masculino , Hipotonia Muscular/complicações , Sequenciamento Completo do Exoma , Adulto Jovem
8.
J Proteome Res ; 13(12): 6135-43, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25311790

RESUMO

Accurate and sensitive detection of protein-protein and protein-RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo. Using quantitative mass spectrometry and deep sequencing, we show that IPL correctly identifies known protein-protein and protein-RNA interactions in the nucleus of mammalian cells. Thus, IPL provides additional temporal and spatial information for the characterization of biological interactions in vivo.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Coloração e Rotulagem/métodos , Biotina/química , Biotina/metabolismo , Cromatografia Líquida , Proteína Potenciadora do Homólogo 2 de Zeste , Células HEK293 , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , Proteínas de Ligação a RNA/química , Análise de Sequência de RNA/métodos , Estreptavidina/química , Estreptavidina/metabolismo , Espectrometria de Massas em Tandem
9.
Genes Dev ; 27(6): 639-53, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23468428

RESUMO

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1-3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle.


Assuntos
Ciclo Celular/fisiologia , Instabilidade Genômica , Sirtuína 2/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Transformação Celular Neoplásica/genética , Cromatina/metabolismo , Dano ao DNA/genética , Técnicas de Inativação de Genes , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Metilação , Camundongos , Camundongos Knockout , Mitose , Ligação Proteica , Sirtuína 2/genética
10.
Genes Dev ; 26(23): 2580-9, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23152447

RESUMO

PR-Set7 is the sole monomethyltransferase responsible for H4K20 monomethylation (H4K20me1) that is the substrate for further methylation by Suv4-20h1/h2. PR-Set7 is required for proper cell cycle progression and is subject to degradation by the CRL4(Cdt2) ubiquitin ligase complex as a function of the cell cycle and DNA damage. This report demonstrates that PR-Set7 is an important downstream effector of CRL4(Cdt2) function during origin of DNA replication licensing, dependent on Suv4-20h1/2 activity. Aberrant rereplication correlates with decreased levels of H4K20me1 and increased levels of H4K20 trimethylation (H4K20me3). Expression of a degradation-resistant PR-Set7 mutant in the mouse embryo that is normally devoid of Suv4-20 does not compromise development or cell cycle progression unless Suv4-20h is coexpressed. PR-Set7 targeting to an artificial locus results in recruitment of the origin recognition complex (ORC) in a manner dependent on Suv4-20h and H4K20me3. Consistent with this, H4K20 methylation status plays a direct role in recruiting ORC through the binding properties of ORC1 and ORCA/LRWD1. Thus, coordinating the status of H4K20 methylation is pivotal for the proper selection of DNA replication origins in higher eukaryotes.


Assuntos
Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , Replicação do DNA/genética , Embrião de Mamíferos , Fibroblastos/citologia , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Ubiquitina-Proteína Ligases/metabolismo
11.
Cell ; 151(1): 181-93, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-23021224

RESUMO

Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.


Assuntos
Células-Tronco Embrionárias/metabolismo , Código das Histonas , Histonas/metabolismo , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Linhagem Celular , Fibroblastos/metabolismo , Células HeLa , Histonas/química , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional
12.
Genes Dev ; 26(4): 325-37, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22345514

RESUMO

Histone post-translational modifications impact many aspects of chromatin and nuclear function. Histone H4 Lys 20 methylation (H4K20me) has been implicated in regulating diverse processes ranging from the DNA damage response, mitotic condensation, and DNA replication to gene regulation. PR-Set7/Set8/KMT5a is the sole enzyme that catalyzes monomethylation of H4K20 (H4K20me1). It is required for maintenance of all levels of H4K20me, and, importantly, loss of PR-Set7 is catastrophic for the earliest stages of mouse embryonic development. These findings have placed PR-Set7, H4K20me, and proteins that recognize this modification as central nodes of many important pathways. In this review, we discuss the mechanisms required for regulation of PR-Set7 and H4K20me1 levels and attempt to unravel the many functions attributed to these proteins.


Assuntos
Ciclo Celular/fisiologia , Cromossomos/metabolismo , Regulação da Expressão Gênica , Genoma , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Animais , Dano ao DNA , Humanos , Lisina/metabolismo , Metilação
13.
Science ; 332(6025): 99-103, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21454787

RESUMO

The carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase 1 (CARM1). Although methylation at R1810 is present on the hyperphosphorylated form of RNAPII in vivo, Ser2 or Ser5 phosphorylation inhibits CARM1 activity toward this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the misexpression of a variety of small nuclear RNAs and small nucleolar RNAs, an effect that is also observed in Carm1(-/-) mouse embryo fibroblasts. These results demonstrate that CTD methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNAPII-associated machinery recruited to distinct gene types.


Assuntos
RNA Polimerase II/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular , Células HeLa , Humanos , Metilação , Camundongos , Mutação , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Polimerase II/genética , RNA Nuclear Pequeno/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas Recombinantes
14.
Mol Cell ; 40(3): 364-76, 2010 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-21035370

RESUMO

The histone methyltransferase PR-Set7/Set8 is the sole enzyme that catalyzes monomethylation of histone H4 at K20 (H4K20me1). Previous reports document disparate evidence regarding PR-Set7 expression during the cell cycle, the biological relevance of PR-Set7 interaction with PCNA, and its role in the cell. We find that PR-Set7 is indeed undetectable during S phase and instead is detected during late G2, mitosis, and early G1. PR-Set7 is transiently recruited to laser-induced DNA damage sites through its interaction with PCNA, after which 53BP1 is recruited dependent on PR-Set7 catalytic activity. During the DNA damage response, PR-Set7 interaction with PCNA through a specialized "PIP degron" domain targets it for PCNA-coupled CRL4(Cdt2)-dependent proteolysis. PR-Set7 mutant in its "PIP degron" is now detectable during S phase, during which the mutant protein accumulates. Outside the chromatin context, Skp2 promotes PR-Set7 degradation as well. These findings demonstrate a stringent spatiotemporal control of PR-Set7 that is essential for preserving the genomic integrity of mammalian cells.


Assuntos
Proteínas Culina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Animais , Biocatálise/efeitos da radiação , Linhagem Celular Tumoral , Ativação Enzimática/efeitos da radiação , Estabilidade Enzimática , Histona-Lisina N-Metiltransferase/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Estrutura Terciária de Proteína , Fase S/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina/metabolismo , Raios Ultravioleta
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