RESUMO
AIM: To determine the impact of a functional human islet clock on insulin secretion and gene transcription. METHODS: Efficient circadian clock disruption was achieved in human pancreatic islet cells by small interfering RNA-mediated knockdown of CLOCK. Human islet secretory function was assessed in the presence or absence of a functional circadian clock by stimulated insulin secretion assays, and by continuous around-the-clock monitoring of basal insulin secretion. Large-scale transcription analysis was accomplished by RNA sequencing, followed by quantitative RT-PCR analysis of selected targets. RESULTS: Circadian clock disruption resulted in a significant decrease in both acute and chronic glucose-stimulated insulin secretion. Moreover, basal insulin secretion by human islet cells synchronized in vitro exhibited a circadian pattern, which was perturbed upon clock disruption. RNA sequencing analysis suggested alterations in 352 transcript levels upon circadian clock disruption. Among them, key regulators of the insulin secretion pathway (GNAQ, ATP1A1, ATP5G2, KCNJ11) and transcripts required for granule maturation and release (VAMP3, STX6, SLC30A8) were affected. CONCLUSIONS: Using our newly developed experimental approach for efficient clock disruption in human pancreatic islet cells, we show for the first time that a functional ß-cell clock is required for proper basal and stimulated insulin secretion. Moreover, clock disruption has a profound impact on the human islet transcriptome, in particular, on the genes involved in insulin secretion.
Assuntos
Proteínas CLOCK/metabolismo , Relógios Circadianos , Hiperglicemia/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas CLOCK/antagonistas & inibidores , Proteínas CLOCK/genética , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Relógios Circadianos/efeitos dos fármacos , Colforsina/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Interferência de RNA , RNA Interferente Pequeno , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteína 3 Associada à Membrana da Vesícula/antagonistas & inibidores , Proteína 3 Associada à Membrana da Vesícula/química , Proteína 3 Associada à Membrana da Vesícula/genética , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Transportador 8 de ZincoRESUMO
Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Cromatina/química , Variação Genética , Genoma Humano , Humanos , Locos de Características Quantitativas , Elementos Reguladores de TranscriçãoRESUMO
OBJECTIVE: To develop and compare two new technologies for diagnosing a contiguous gene syndrome, the Williams-Beuren syndrome (WBS). METHODS: The first proposed method, named paralogous sequence quantification (PSQ), is based on the use of paralogous sequences located on different chromosomes and quantification of specific mismatches present at these loci using pyrosequencing technology. The second exploits quantitative real time polymerase chain reaction (QPCR) to assess the relative quantity of an analysed locus. RESULTS: A correct and unambiguous diagnosis was obtained for 100% of the analysed samples with either technique (n = 165 and n = 155, respectively). These methods allowed the identification of two patients with atypical deletions in a cohort of 182 WBS patients. Both patients presented with mild facial anomalies, mild mental retardation with impaired visuospatial cognition, supravalvar aortic stenosis, and normal growth indices. These observations are consistent with the involvement of GTF2IRD1 or GTF2I in some of the WBS facial features. CONCLUSIONS: Both PSQ and QPCR are robust, easy to interpret, and simple to set up. They represent a competitive alternative for the diagnosis of segmental aneuploidies in clinical laboratories. They have advantages over fluorescence in situ hybridisation or microsatellites/SNP genotyping for detecting short segmental aneuploidies as the former is costly and labour intensive while the latter depends on the informativeness of the polymorphisms.
Assuntos
Aneuploidia , Síndrome de Williams/genética , Diagnóstico Diferencial , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , Polimorfismo Genético , Deleção de Sequência , Síndrome de Williams/classificação , Síndrome de Williams/diagnósticoRESUMO
BACKGROUND: Chromosomal aneuploidies are a common cause of congenital disorders associated with cognitive impairment and multiple dysmorphic features. Pre-natal diagnosis of aneuploidies is most commonly performed by the karyotyping of fetal cells obtained by amniocentesis or chorionic villus sampling, but this method is labour intensive and requires about 14 days to complete. METHODS: We have developed a PCR based method for the detection of targeted chromosome number abnormalities termed paralogous sequence quantification (PSQ), based on the use of paralogous genes. Paralogous sequences have a high degree of sequence identity, but accumulate nucleotide substitutions in a locus specific manner. These sequence differences, which we term paralogous sequence mismatches (PSMs), can be quantified using pyrosequencing technology, to estimate the relative dosage between different chromosomes. We designed 10 assays for the detection of trisomies of chromosomes 13, 18, and 21 and sex chromosome aneuploidies. RESULTS: We evaluated the performance of this method on 175 DNAs, highly enriched for abnormal samples. A correct and unambiguous diagnosis was given for 119 out of 120 aneuploid samples as well as for all the controls. One sample which gave an intermediate value for the chromosome 13 assays could not be diagnosed. CONCLUSIONS: Our data suggests that PSQ is a robust, easy to interpret, and easy to set up method for the diagnosis of common aneuploidies, and can be performed in less than 48 h, representing a competitive alternative for widespread use in diagnostic laboratories.
Assuntos
Aneuploidia , Testes Genéticos/métodos , Reação em Cadeia da Polimerase/métodos , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 21 , Cromossomos Humanos X , Cromossomos Humanos Y , DNA , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Homologia de Sequência do Ácido NucleicoRESUMO
In this article we show, using scanning tunneling spectroscopy, the existence of static striped density of electronic states in nearly optimally doped Bi2Sr2CaCu2O8+delta in zero field. This modulation is aligned with the Cu-O bonds, with a periodicity of four lattice constants, and exhibits features characteristic of a two-dimensional system of line objects. We further show that the density of states modulation manifests itself as a shift of states from above to below the superconducting gap. The fact that a single energy scale (i.e., the gap) appears for both superconductivity and stripes suggests that these two effects have the same origin.