RESUMEN
Dynamic cell identities underlie flexible developmental programs. The stomatal lineage in the Arabidopsis leaf epidermis features asynchronous and indeterminate divisions that can be modulated by environmental cues. The products of the lineage, stomatal guard cells and pavement cells, regulate plant-atmosphere exchanges, and the epidermis as a whole influences overall leaf growth. How flexibility is encoded in development of the stomatal lineage and how cell fates are coordinated in the leaf are open questions. Here, by leveraging single-cell transcriptomics and molecular genetics, we uncovered models of cell differentiation within Arabidopsis leaf tissue. Profiles across leaf tissues identified points of regulatory congruence. In the stomatal lineage, single-cell resolution resolved underlying cell heterogeneity within early stages and provided a fine-grained profile of guard cell differentiation. Through integration of genome-scale datasets and spatiotemporally precise functional manipulations, we also identified an extended role for the transcriptional regulator SPEECHLESS in reinforcing cell fate commitment.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Hojas de la Planta/crecimiento & desarrollo , Estomas de Plantas/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Linaje de la Célula , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Estomas de Plantas/citología , RNA-Seq , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by a uniform 1.5-Mb duplication on chromosome 17p, which includes the PMP22 gene. Patients often present the classic neuropathy phenotype, but also with high clinical variability. OBJECTIVE: We aimed to identify genetic variants that are potentially associated with specific clinical outcomes in CMT1A. METHODS: We genotyped over 600,000 genomic markers using DNA samples from 971 CMT1A patients and performed a case-only genome-wide association study (GWAS) to identify potential genetic association in a subset of 644 individuals of European ancestry. A total of 14 clinical outcomes were analyzed in this study. RESULTS: The analyses yielded suggestive association signals in four clinical outcomes: difficulty with eating utensils (lead SNP rs4713376, chr6â:â30773314, Pâ=â9.91×10-7, odds ratioâ=â3.288), hearing loss (lead SNP rs7720606, chr5â:â126551732, Pâ=â2.08×10-7, odds ratioâ=â3.439), decreased ability to feel (lead SNP rs17629990, chr4â:â171224046, Pâ=â1.63×10-7, odds ratioâ=â0.336), and CMT neuropathy score (lead SNP rs12137595, chr1â:â4094068, Pâ=â1.14×10-7, betaâ=â3.014). CONCLUSIONS: While the results require validation in future genetic and functional studies, the detected association signals may point to novel genetic modifiers in CMT1A.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Genes Modificadores/genética , Estudio de Asociación del Genoma Completo , Genotipo , HumanosRESUMEN
OBJECTIVE: Genetic modifiers in rare disease have long been suspected to contribute to the considerable variance in disease expression, including Charcot-Marie-Tooth disease type 1A (CMT1A). To address this question, the Inherited Neuropathy Consortium collected a large standardized sample of such rare CMT1A patients over a period of 8 years. CMT1A is caused in most patients by a uniformly sized 1.5 Mb duplication event involving the gene PMP22. METHODS: We genotyped DNA samples from 971 CMT1A patients on Illumina BeadChips. Genome-wide analysis was performed in a subset of 330 of these patients, who expressed the extremes of a hallmark symptom: mild and severe foot dorsiflexion strength impairment. SIPA1L2 (signal-induced proliferation-associated 1 like 2), the top identified candidate modifier gene, was expressed in the peripheral nerve, and our functional studies identified and confirmed interacting proteins using coimmunoprecipitation analysis, mass spectrometry, and immunocytochemistry. Chromatin immunoprecipitation and in vitro siRNA experiments were used to analyze gene regulation. RESULTS: We identified significant association of 4 single nucleotide polymorphisms (rs10910527, rs7536385, rs4649265, rs1547740) in SIPA1L2 with foot dorsiflexion strength (p < 1 × 10-7 ). Coimmunoprecipitation and mass spectroscopy studies identified ß-actin and MYH9 as SIPA1L2 binding partners. Furthermore, we show that SIPA1L2 is part of a myelination-associated coexpressed network regulated by the master transcription factor SOX10. Importantly, in vitro knockdown of SIPA1L2 in Schwannoma cells led to a significant reduction of PMP22 expression, hinting at a potential strategy for drug development. INTERPRETATION: SIPA1L2 is a potential genetic modifier of CMT1A phenotypic expressions and offers a new pathway to therapeutic interventions. ANN NEUROL 2019;85:316-330.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Pie/fisiopatología , Proteínas Activadoras de GTPasa/genética , Genes Modificadores/genética , Debilidad Muscular/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Niño , Preescolar , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Debilidad Muscular/fisiopatología , Proteínas de la Mielina/genética , Neurilemoma/genética , Fenotipo , Polimorfismo de Nucleótido Simple , Ratas , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 (NRG1) type III regulate Schwann cell fate and myelination. Here we ask if modulating NRG1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we improved the myelination defects by early overexpression of NRG1 type III. Surprisingly, the improvement was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 and oligodendrocyte myelin glycoprotein. We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial NRG1 type III signaling has beneficial effects and improves myelination defects during development in a model of CHN.
Asunto(s)
Vaina de Mielina/metabolismo , Neurregulina-1/genética , Neurregulina-1/fisiología , Potenciales de Acción , Animales , Axones/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen/métodos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/genética , Neurregulina-1/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Nervios Periféricos/metabolismo , Células de Schwann/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Measurement of gene expression for high-throughput screening is an increasingly used technique that has been developed for not only gene dosage disorders resulting from disease-associated copy number variations, but also for induction/repression of genes modulating the severity of a disease phenotype. Traditional methods have employed transient or stable transfection of reporter constructs in which a single reporter is driven by selected regulatory elements from the candidate gene. However, individual regulatory elements are inherently unable to capture the integrated regulation of multiple enhancers at the endogenous locus, and random reporter insertion can result in neighborhood effects that impact the physiological responsiveness of the reporter. Therefore, we outline a general method of employing genome editing to insert reporters into the 3' UTR of a candidate gene, which has been used successfully in our studies of the Pmp22 gene associated with Charcot-Marie-Tooth disease. The method employs genome editing to insert two nonhomologous reporters that maximize the efficiency of identification of biologically active molecules through concordant responses in small molecule screening. We include a number of aspects of the design and construction of these reporter assays that will be applicable to creation of similar assays in a variety of cell types.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Edición Génica/métodos , Genes Reporteros/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de la Mielina/genética , Bioensayo/instrumentación , Bioensayo/métodos , Sistemas CRISPR-Cas/genética , Línea Celular , Edición Génica/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Luciferasas de Luciérnaga/química , Luciferasas de Luciérnaga/genética , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodos , Transfección/instrumentación , Transfección/métodosRESUMEN
OBJECTIVE: Our goal was to define the genetic cause of the profound hypomyelination in the taiep rat model and determine its relevance to human white matter disease. METHODS: Based on previous localization of the taiep mutation to rat chromosome 9, we tested whether the mutation resided within the Tubb4a (ß-tubulin 4A) gene, because mutations in the TUBB4A gene have been described in patients with central nervous system hypomyelination. To determine whether accumulation of microtubules led to progressive demyelination, we analyzed the spinal cord and optic nerves of 2-year-old rats by light and electron microscopy. Cerebral white matter from a patient with TUBB4A Asn414Lys mutation and magnetic resonance imaging evidence of severe hypomyelination were studied similarly. RESULTS: As the taiep rat ages, there is progressive loss of myelin in the brain and dorsal column of the spinal cord associated with increased oligodendrocyte numbers with accumulation of microtubules. This accumulation involved the entire cell body and distal processes of oligodendrocytes, but there was no accumulation of microtubules in axons. A single point mutation in Tubb4a (p.Ala302Thr) was found in homozygous taiep samples. A similar hypomyelination associated with increased oligodendrocyte numbers and arrays of microtubules in oligodendrocytes was demonstrated in the human patient sample. INTERPRETATION: The taiep rat is the first animal model of TUBB4 mutations in humans and a novel system in which to test the mechanism of microtubule accumulation. The finding of microtubule accumulation in a patient with a TUBB4A mutation and leukodystrophy confirms the usefulness of taiep as a model of the human disease. Ann Neurol 2017;81:690-702.
Asunto(s)
Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Microtúbulos/metabolismo , Nervio Óptico/diagnóstico por imagen , Médula Espinal/diagnóstico por imagen , Tubulina (Proteína)/genética , Sustancia Blanca/diagnóstico por imagen , Animales , Preescolar , Enfermedades Desmielinizantes/diagnóstico por imagen , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/fisiopatología , Humanos , Imagen por Resonancia Magnética , Microscopía Electrónica , RatasRESUMEN
Schwann cells and oligodendrocytes are the myelinating cells of the peripheral and central nervous system, respectively. Despite having different myelin components and different transcription factors driving their terminal differentiation there are shared molecular mechanisms between the two. Sox10 is one common transcription factor required for several steps in development of myelinating glia. However, other factors are divergent as Schwann cells need the transcription factor early growth response 2/Krox20 and oligodendrocytes require Myrf. Likewise, some signaling pathways, like the Erk1/2 kinases, are necessary in both cell types for proper myelination. Nonetheless, the molecular mechanisms that control this shared signaling pathway in myelinating cells remain only partially characterized. The hypothesis of this study is that signaling pathways that are similarly regulated in both Schwann cells and oligodendrocytes play central roles in coordinating the differentiation of myelinating glia. To address this hypothesis, we have used genome-wide binding data to identify a relatively small set of genes that are similarly regulated by Sox10 in myelinating glia. We chose one such gene encoding Dual specificity phosphatase 15 (Dusp15) for further analysis in Schwann cell signaling. RNA interference and gene deletion by genome editing in cultured RT4 and primary Schwann cells showed Dusp15 is necessary for full activation of Erk1/2 phosphorylation. In addition, we show that Dusp15 represses expression of several myelin genes, including myelin basic protein. The data shown here support a mechanism by which early growth response 2 activates myelin genes, but also induces a negative feedback loop through Dusp15 to limit over-expression of myelin genes.
Asunto(s)
Fosfatasas de Especificidad Dual/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Vaina de Mielina/enzimología , Células de Schwann/enzimología , Animales , Línea Celular , Activación Enzimática/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/genética , RatasRESUMEN
Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice, we show that Taz is required in Schwann cells for radial sorting and myelination and that Yap is redundant with Taz. Yap and Taz are activated in Schwann cells by mechanical stimuli and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Vaina de Mielina/metabolismo , Fosfoproteínas/metabolismo , Células de Schwann/metabolismo , Factores de Transcripción/metabolismo , Aciltransferasas , Animales , Axones/fisiología , Axones/ultraestructura , Proteínas de Ciclo Celular , Células Cultivadas , Mecanotransducción Celular/fisiología , Ratones Endogámicos C57BL , Neurogénesis/fisiología , Receptores de Laminina/metabolismo , Células de Schwann/citología , Proteínas Señalizadoras YAPRESUMEN
Schwann cells are myelinating glia in the peripheral nervous system that form the myelin sheath. A major cause of peripheral neuropathy is a copy number variant involving the Peripheral Myelin Protein 22 (PMP22) gene, which is located within a 1.4-Mb duplication on chromosome 17 associated with the most common form of Charcot-Marie-Tooth Disease (CMT1A). Rodent models of CMT1A have been used to show that reducing Pmp22 overexpression mitigates several aspects of a CMT1A-related phenotype. Mechanistic studies of Pmp22 regulation identified enhancers regulated by the Sox10 (SRY sex determining region Y-box 10) and Egr2/Krox20 (Early growth response protein 2) transcription factors in myelinated nerves. However, relatively little is known regarding how other transcription factors induce Pmp22 expression during Schwann cell development and myelination. Here, we examined Pmp22 enhancers as a function of cell type-specificity, nerve injury and development. While Pmp22 enhancers marked by active histone modifications were lost or remodeled after injury, we found that these enhancers were permissive in early development prior to Pmp22 upregulation. Pmp22 enhancers contain binding motifs for TEA domain (Tead) transcription factors of the Hippo signaling pathway. We discovered that Tead1 and co-activators Yap and Taz are required for Pmp22 expression, as well as for the expression of Egr2 Tead1 directly binds Pmp22 and Egr2 enhancers early in development and Tead1 binding is induced during myelination, correlating with Pmp22 expression. The data identify Tead1 as a novel regulator of Pmp22 expression during development in concert with Sox10 and Egr2.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Proteínas de Unión al ADN/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteínas de la Mielina/genética , Enfermedades del Sistema Nervioso Periférico/genética , Factores de Transcripción SOXE/genética , Factores de Transcripción/genética , Animales , Enfermedad de Charcot-Marie-Tooth/patología , Variaciones en el Número de Copia de ADN/genética , Proteínas de Unión al ADN/biosíntesis , Modelos Animales de Enfermedad , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Regulación de la Expresión Génica/genética , Humanos , Ratones , Vaina de Mielina/genética , Vaina de Mielina/patología , Neurogénesis/genética , Enfermedades del Sistema Nervioso Periférico/patología , Fenotipo , Células de Schwann/metabolismo , Células de Schwann/patología , Factores de Transcripción de Dominio TEA , Factores de Transcripción/biosíntesisRESUMEN
Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. GLIA 2015;63:1897-1914.
RESUMEN
As next generation sequencing technologies are becoming more economical, large-scale ChIP-seq studies are enabling the investigation of the roles of transcription factor binding and epigenome on phenotypic variation. Studying such variation requires individual level ChIP-seq experiments. Standard designs for ChIP-seq experiments employ a paired control per ChIP-seq sample. Genomic coverage for control experiments is often sacrificed to increase the resources for ChIP samples. However, the quality of ChIP-enriched regions identifiable from a ChIP-seq experiment depends on the quality and the coverage of the control experiments. Insufficient coverage leads to loss of power in detecting enrichment. We investigate the effect of in silico pooling of control samples within multiple biological replicates, multiple treatment conditions, and multiple cell lines and tissues across multiple datasets with varying levels of genomic coverage. Our computational studies suggest guidelines for performing in silico pooling of control experiments. Using vast amounts of ENCODE data, we show that pairwise correlations between control samples originating from multiple biological replicates, treatments, and cell lines/tissues can be grouped into two classes representing whether or not in silico pooling leads to power gain in detecting enrichment between the ChIP and the control samples. Our findings have important implications for multiplexing samples.
Asunto(s)
Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Simulación por Computador , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Factor de Unión a CCCTC , Análisis por Conglomerados , Histona Desacetilasas/genética , Histonas/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Células K562 , Ratas , Proteínas Represoras/genéticaRESUMEN
The structural integrity of myelin formed by Schwann cells in the peripheral nervous system (PNS) is required for proper nerve conduction and is dependent on adequate expression of myelin genes including peripheral myelin protein 22 (PMP22). Consequently, excess PMP22 resulting from its genetic duplication and overexpression has been directly associated with the peripheral neuropathy called Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent type of CMT. Here, in an attempt to identify transcriptional inhibitors with therapeutic value toward CMT1A, we developed a cross-validating pair of orthogonal reporter assays, firefly luciferase (FLuc) and ß-lactamase (ßLac), capable of recapitulating PMP22 expression, utilizing the intronic regulatory element of the human PMP22 gene. Each compound from a collection of approximately 3,000 approved drugs was tested at multiple titration points to achieve a pharmacological end point in a 1536-well plate quantitative high-throughput screen (qHTS) format. In conjunction with an independent counter-screen for cytotoxicity, the design of our orthogonal screen platform effectively contributed to selection and prioritization of active compounds, among which three drugs (fenretinide, olvanil, and bortezomib) exhibited marked reduction of endogenous Pmp22 mRNA and protein. Overall, the findings of this study provide a strategic approach to assay development for gene-dosage diseases such as CMT1A.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Sistemas de Liberación de Medicamentos/métodos , Dosificación de Gen/fisiología , Marcación de Gen/métodos , Proteínas de la Mielina/antagonistas & inhibidores , Proteínas de la Mielina/genética , Capsaicina/administración & dosificación , Capsaicina/análogos & derivados , Enfermedad de Charcot-Marie-Tooth/tratamiento farmacológico , Enfermedad de Charcot-Marie-Tooth/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Fenretinida/administración & dosificación , Dosificación de Gen/efectos de los fármacos , Humanos , Proteínas de la Mielina/biosíntesisRESUMEN
Schwann cell differentiation and subsequent myelination of the peripheral nervous system require the action of several transcription factors, including Sox10, which is vital at multiple stages of development. The transition from immature to myelinating Schwann cell is also regulated posttranscriptionally and depends upon Dicer-mediated processing of microRNAs (miRNAs). Although specific miRNA targets have begun to be identified, the mechanisms establishing the dynamic regulation of miRNA expression have not been elucidated. We performed expression profiling studies and identified 225 miRNAs differentially expressed during peripheral myelination. A subset of 9 miRNAs is positively regulated by Sox10, including miR-338 which has been implicated in oligodendrocyte maturation. In vivo chromatin immunoprecipitation (ChIP) of sciatic nerve cells revealed a Sox10 binding site upstream of an alternate promoter within the Aatk gene, which hosts miR-338. Sox10 occupied this site in spinal cord ChIP experiments, suggesting a similar regulatory mechanism in oligodendrocytes. Cancer profiling studies have identified clusters of miRNAs that regulate proliferation, termed "oncomirs." In Schwann cells, the expression of many of these proproliferative miRNAs was reduced in the absence of Sox10. Finally, Schwann cells with reduced Sox10 and oncomir expression have an increase in the CDK inhibitor p21 and a concomitant reduction in cell proliferation.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Células de Schwann/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Secuencia de Bases , Línea Celular , Proliferación Celular , Células Cultivadas , Humanos , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Células de Schwann/citología , Nervio Ciático/citologíaRESUMEN
The early growth response (EGR) family of transcription factors has been implicated in control of lipid biosynthetic genes. Egr1 is induced by insulin both in vitro and in vivo and is the most highly expressed family member in liver. In this study, we investigated whether Egr1 regulates cholesterol biosynthetic genes in liver. Using an insulin-sensitive liver cell line, we show that localization of Egr1 to cholesterol biosynthetic genes is induced by insulin treatment and that this localization precedes the induction of the genes. Reduction in Egr1 expression using targeted siRNA blunted the insulin-dependent induction of cholesterol genes. A similar reduction in squalene epoxidase expression was also observed in Egr1 null mice. In addition, application of chromatin immunoprecipitation (ChIP) samples to tiled gene microarrays revealed localization of Egr1 in promoter regions of many cholesterol gene loci. In vivo ChIP assays using liver tissue show that Egr1 localization to several cholesterol biosynthetic gene promoters is induced by feeding. Finally, analysis of plasma cholesterol in Egr1(-/-) mice indicated a significant decrease in serum cholesterol when compared with wild-type mice. Together these data point to Egr1 as a modulator of the cholesterol biosynthetic gene family in liver.
Asunto(s)
Colesterol/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación de la Expresión Génica/fisiología , Hígado/metabolismo , Regiones Promotoras Genéticas/fisiología , Animales , Colesterol/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Ratones , Ratones Mutantes , RatasRESUMEN
Successful myelination of the peripheral nervous system depends upon induction of major protein components of myelin, such as peripheral myelin protein 22 (PMP22). Myelin stability is also sensitive to levels of PMP22, as a 1.4 Mb duplication on human chromosome 17, resulting in three copies of PMP22, is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth disease. The transcription factor Egr2/Krox20 is required for induction of high level expression of Pmp22 in Schwann cells but its activation elements have not yet been determined. Using chromatin immunoprecipitation analysis of the rat Pmp22 locus, we found a major peak of Egr2 binding within the large intron of the Pmp22 gene. Analysis of a 250 bp region within the largest intron showed that it is strongly activated by Egr2 expression in reporter assays. Moreover, this region contains conserved binding sites not only for Egr2 but also for Sox10, which is also required for Schwann cell development. Our analysis shows that Sox10 is required for optimal activity of the intronic site as well as PMP22 expression. Finally, mouse transgenic analysis revealed tissue-specific expression of this intronic sequence in peripheral nerve. Overall, these data show that Egr2 and Sox10 activity are directly involved in mediating the developmental induction of Pmp22 expression.