RESUMO
Peripheral nerve myelination is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot-Marie-Tooth Disease. This form, classified as CMT1A, is caused by a 1.4 Mb duplication on chromosome 17, which includes the abundantly expressed Schwann cell myelin gene, Peripheral Myelin Protein 22 (PMP22). This is one of the most common copy number variants causing neurological disease. Overexpression of Pmp22 in rodent models recapitulates several aspects of neuropathy, and reduction of Pmp22 in such models results in amelioration of the neuropathy phenotype. Recently we identified a potential super-enhancer approximately 90-130 kb upstream of the Pmp22 transcription start sites. This super-enhancer encompasses a cluster of individual enhancers that have the acetylated histone H3K27 active enhancer mark, and coincides with smaller duplications identified in patients with milder CMT1A-like symptoms, where the PMP22 coding region itself was not part of the duplication. In this study, we have utilized genome editing to create a deletion of this super-enhancer to determine its role in Pmp22 regulation. Our data show a significant decrease in Pmp22 transcript expression using allele-specific internal controls. Moreover, the P2 promoter of the Pmp22 gene, which is used in other cell types, is affected, but we find that the Schwann cell-specific P1 promoter is disproportionately more sensitive to loss of the super-enhancer. These data show for the first time the requirement of these upstream enhancers for full Pmp22 expression.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Variações do Número de Cópias de DNA/genética , Elementos Facilitadores Genéticos/genética , Proteínas da Mielina/genética , Animais , Linhagem Celular , Doença de Charcot-Marie-Tooth/patologia , Duplicação Cromossômica/genética , Cromossomos Humanos Par 17/genética , Edição de Genes , Regulação da Expressão Gênica/genética , Humanos , Regiões Promotoras Genéticas , Ratos , Células de Schwann/patologiaRESUMO
UNLABELLED: The rapid and dynamic transcriptional changes of Schwann cells in response to injury are critical to peripheral nerve repair, yet the epigenomic reprograming that leads to the induction of injury-activated genes has not been characterized. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3), which produces a transcriptionally repressive chromatin environment. We find that many promoters and/or gene bodies of injury-activated genes of mature rat nerves are occupied with H3K27me3. In contrast, the majority of distal enhancers that gain H3K27 acetylation after injury are not repressed by H3K27 methylation before injury, which is normally observed in developmentally poised enhancers. Injury induces demethylation of H3K27 in many genes, such as Sonic hedgehog (Shh), which is silenced throughout Schwann cell development before injury. In addition, experiments using a Schwann cell-specific mouse knock-out of the Eed subunit of PRC2 indicate that demethylation is a rate-limiting step in the activation of such genes. We also show that some transcription start sites of H3K27me3-repressed injury genes of uninjured nerves are bound with a mark of active promoters H3K4me3, for example, Shh and Gdnf, and the reduction of H3K27me3 results in increased trimethylation of H3K4. Our findings identify reversal of polycomb repression as a key step in gene activation after injury. SIGNIFICANCE STATEMENT: Peripheral nerve regeneration after injury is dependent upon implementation of a novel genetic program in Schwann cells that supports axonal survival and regeneration. Identifying means to enhance Schwann cell reprogramming after nerve injury could be used to foster effective remyelination in the treatment of demyelinating disorders and in identifying pathways involved in regenerative process of myelination. Although recent progress has identified transcriptional determinants of successful reprogramming of the Schwann cell transcriptome after nerve injury, our results have highlighted a novel epigenomic pathway in which reversal of the Polycomb pathway of repressive histone methylation is required for activation of a significant number of injury-induced genes.
Assuntos
Reprogramação Celular/fisiologia , Epigenômica , Regulação da Expressão Gênica/fisiologia , Células de Schwann/metabolismo , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Animais , Benzazepinas/farmacologia , Reprogramação Celular/genética , Imunoprecipitação da Cromatina , Biologia Computacional , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Histonas/genética , Histonas/metabolismo , Técnicas In Vitro , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração/genética , Células de Schwann/efeitos dos fármacos , Células de Schwann/fisiologia , Transdução de Sinais/genéticaRESUMO
Myelination of peripheral nerves by Schwann cells requires coordinate regulation of gene repression as well as gene activation. Several chromatin remodeling pathways critical for peripheral nerve myelination have been identified, but the functions of histone methylation in the peripheral nerve have not been elucidated. To determine the role of histone H3 Lys27 methylation, we have generated mice with a Schwann cell-specific knock-out of Eed, which is an essential subunit of the polycomb repressive complex 2 (PRC2) that catalyzes methylation of histone H3 Lys27. Analysis of this mutant revealed no significant effects on early postnatal development of myelin. However, its loss eventually causes progressive hypermyelination of small-diameter axons and apparent fragmentation of Remak bundles. These data identify the PRC2 complex as an epigenomic modulator of mature myelin thickness, which is associated with changes in Akt phosphorylation. Interestingly, we found that Eed inactivation causes derepression of several genes, e.g., Sonic hedgehog (Shh) and Insulin-like growth factor-binding protein 2 (Igfbp2), that become activated after nerve injury, but without activation of a primary regulator of the injury program, c-Jun. Analysis of the activated genes in cultured Schwann cells showed that Igfbp2 regulates Akt activation. Our results identify an epigenomic pathway required for establishing thickness of mature myelin and repressing genes that respond to nerve injury.
Assuntos
Regulação da Expressão Gênica/fisiologia , Bainha de Mielina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Hedgehog/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Proteína P0 da Mielina/genética , Proteína Oncogênica v-akt/metabolismo , Complexo Repressor Polycomb 2/genética , Ratos , Nervo Isquiático/ultraestrutura , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Myelination of the peripheral nervous system is required for axonal function and long term stability. After peripheral nerve injury, Schwann cells transition from axon myelination to a demyelinated state that supports neuronal survival and ultimately remyelination of axons. Reprogramming of gene expression patterns during development and injury responses is shaped by the actions of distal regulatory elements that integrate the actions of multiple transcription factors. We used ChIP-seq to measure changes in histone H3K27 acetylation, a mark of active enhancers, to identify enhancers in myelinating rat peripheral nerve and their dynamics after demyelinating nerve injury. Analysis of injury-induced enhancers identified enriched motifs for c-Jun, a transcription factor required for Schwann cells to support nerve regeneration. We identify a c-Jun-bound enhancer in the gene for Runx2, a transcription factor induced after nerve injury, and we show that Runx2 is required for activation of other induced genes. In contrast, enhancers that lose H3K27ac after nerve injury are enriched for binding sites of the Sox10 and early growth response 2 (Egr2/Krox20) transcription factors, which are critical determinants of Schwann cell differentiation. Egr2 expression is lost after nerve injury, and many Egr2-binding sites lose H3K27ac after nerve injury. However, the majority of Egr2-bound enhancers retain H3K27ac, indicating that other transcription factors maintain active enhancer status after nerve injury. The global epigenomic changes in H3K27ac deposition pinpoint dynamic changes in enhancers that mediate the effects of transcription factors that control Schwann cell myelination and peripheral nervous system responses to nerve injury.
Assuntos
Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Células de Schwann/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Doenças Desmielinizantes/complicações , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Bainha de Mielina/genética , Bainha de Mielina/patologia , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/patologia , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
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.
RESUMO
The molecular basis of the organogenesis, homeostasis, and tumorigenesis of the adrenal cortex has been the subject of intense study for many decades. Specifically, characterization of tumor predisposition syndromes with adrenocortical manifestations and molecular profiling of sporadic adrenocortical tumors have led to the discovery of key molecular pathways that promote pathological adrenal growth. However, given the observational nature of such studies, several important questions regarding the molecular pathogenesis of adrenocortical tumors have remained. This review will summarize naturally occurring and genetically engineered mouse models that have provided novel tools to explore the molecular and cellular underpinnings of adrenocortical tumors. New paradigms of cancer initiation, maintenance, and progression that have emerged from this work will be discussed.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , Modelos Animais de Doenças , Neoplasias do Córtex Suprarrenal/metabolismo , Animais , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Proteína Supressora de Tumor p53/genética , Via de Sinalização WntRESUMO
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.