RESUMEN
Inborn defects in DNA repair are associated with complex developmental disorders whose causal mechanisms are poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the nucleotide excision repair (NER) structure-specific endonuclease ERCC1-XPF complex interacts with the insulator binding protein CTCF, the cohesin subunits SMC1A and SMC3 and with MBD2; the factors co-localize with ATRX at the promoters and control regions (ICRs) of imprinted genes during postnatal hepatic development. Loss of Ercc1 or exposure to MMC triggers the localization of CTCF to heterochromatin, the dissociation of the CTCF-cohesin complex and ATRX from promoters and ICRs, altered histone marks and the aberrant developmental expression of imprinted genes without altering DNA methylation. We propose that ERCC1-XPF cooperates with CTCF and cohesin to facilitate the developmental silencing of imprinted genes and that persistent DNA damage triggers chromatin changes that affect gene expression programs associated with NER disorders.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Silenciador del Gen , Impresión Genómica , Proteínas Represoras/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas Cromosómicas no Histona/genética , Técnicas de Cocultivo , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Fibroblastos/enzimología , Regulación del Desarrollo de la Expresión Génica , Genotipo , Histonas/metabolismo , Hígado/enzimología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteína Nuclear Ligada al Cromosoma X , CohesinasRESUMEN
BACKGROUND: Brain derived neurotrophic factor (BDNF) belongs to a family of structurally related proteins called neurotrophins that have been shown to regulate survival and growth of neurons in the developing central and peripheral nervous system and also to take part in synaptic plasticity related processes in adulthood. Since BDNF is associated with several nervous system disorders it would be beneficial to have cellular reporter system for studying its expression regulation. METHODS: Using modified bacterial artificial chromosome (BAC), we generated several transgenic cell lines expressing humanised Renilla luciferase (hRluc)-EGFP fusion reporter gene under the control of rat BDNF gene regulatory sequences (rBDNF-hRluc-EGFP) in HeLa background. To see if the hRluc-EGFP reporter was regulated in response to known regulators of BDNF expression we treated cell lines with substances known to regulate BDNF and also overexpressed transcription factors known to regulate BDNF gene in established cell lines. RESULTS: rBDNF-hRluc-EGFP cell lines had high transgene copy numbers when assayed with qPCR and FISH analysis showed that transgene was maintained episomally in all cell lines. Luciferase activity in transgenic cell lines was induced in response to ionomycin-mediated rise of intracellular calcium levels, treatment with HDAC inhibitors and by over-expression of transcription factors known to increase BDNF expression, indicating that transcription of the transgenic reporter is regulated similarly to the endogenous BDNF gene. CONCLUSIONS: Generated rBDNF-hRluc-EGFP BAC cell lines respond to known modulators of BDNF expression and could be used for screening of compounds/small molecules or transcription factors altering BDNF expression.
Asunto(s)
Bioensayo/métodos , Factor Neurotrófico Derivado del Encéfalo/genética , Cromosomas Artificiales Bacterianos/genética , Diseño de Fármacos , Inhibidores de Histona Desacetilasas/farmacología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Células HeLa , HumanosRESUMEN
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of neurotrophic factors, has important functions in the peripheral and central nervous system of vertebrates. We have generated bacterial artificial chromosome (BAC) transgenic mice harboring 207 kb of the rat BDNF (rBDNF) locus containing the gene, 13 kb of genomic sequences upstream of BDNF exon I, and 144 kb downstream of protein encoding exon IX, in which protein coding region was replaced with the lacZ reporter gene. This BDNF-BAC drove transgene expression in the brain, heart, and lung, recapitulating endogenous BDNF expression to a larger extent than shorter rat BDNF transgenes employed previously. Moreover, kainic acid induced the expression of the transgenic BDNF mRNA in the cerebral cortex and hippocampus through preferential activation of promoters I and IV, thus recapitulating neuronal activity-dependent transcription of the endogenous BDNF gene.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Cromosomas Artificiales Bacterianos/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/fisiología , Transgenes , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Corteza Cerebral/metabolismo , Cromosomas Artificiales Bacterianos/genética , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Ácido Kaínico/farmacología , Ratones , Ratones Transgénicos , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , RatasRESUMEN
BACKGROUND: Alterations in brain-derived neurotrophic factor (BDNF) gene expression contribute to serious pathologies such as depression, epilepsy, cancer, Alzheimer's, Huntington and Parkinson's disease. Therefore, exploring the mechanisms of BDNF regulation represents a great clinical importance. Studying BDNF expression remains difficult due to its multiple neural activity-dependent and tissue-specific promoters. Thus, microarray data could provide insight into the regulation of this complex gene. Conventional microarray co-expression analysis is usually carried out by merging the datasets or by confirming the re-occurrence of significant correlations across datasets. However, co-expression patterns can be different under various conditions that are represented by subsets in a dataset. Therefore, assessing co-expression by measuring correlation coefficient across merged samples of a dataset or by merging datasets might not capture all correlation patterns. RESULTS: In our study, we performed meta-coexpression analysis of publicly available microarray data using BDNF as a "guide-gene" introducing a "subset" approach. The key steps of the analysis included: dividing datasets into subsets with biologically meaningful sample content (e.g. tissue, gender or disease state subsets); analyzing co-expression with the BDNF gene in each subset separately; and confirming co- expression links across subsets. Finally, we analyzed conservation in co-expression with BDNF between human, mouse and rat, and sought for conserved over-represented TFBSs in BDNF and BDNF-correlated genes. Correlated genes discovered in this study regulate nervous system development, and are associated with various types of cancer and neurological disorders. Also, several transcription factor identified here have been reported to regulate BDNF expression in vitro and in vivo. CONCLUSION: The study demonstrates the potential of the "subset" approach in co-expression conservation analysis for studying the regulation of single genes and proposes novel regulators of BDNF gene expression.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Algoritmos , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Análisis por Conglomerados , Regulación de la Expresión Génica , Humanos , Ratones , Ratas , Elementos Reguladores de la TranscripciónRESUMEN
BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a small secreted protein that has important roles in the developing and adult nervous system. Altered expression or changes in the regulation of the BDNF gene have been implicated in a variety of human nervous system disorders. Although regulation of the rodent BDNF gene has been extensively investigated, in vivo studies regarding the human BDNF gene are largely limited to postmortem analysis. Bacterial artificial chromosome (BAC) transgenic mice harboring the human BDNF gene and its regulatory flanking sequences constitute a useful tool for studying human BDNF gene regulation and for identification of therapeutic compounds modulating BDNF expression. RESULTS: In this study we have generated and analyzed BAC transgenic mice carrying 168 kb of the human BDNF locus modified such that BDNF coding sequence was replaced with the sequence of a fusion protein consisting of N-terminal BDNF and the enhanced green fluorescent protein (EGFP). The human BDNF-BAC construct containing all BDNF 5' exons preceded by different promoters recapitulated the expression of endogenous BDNF mRNA in the brain and several non-neural tissues of transgenic mice. All different 5' exon-specific BDNF-EGFP alternative transcripts were expressed from the transgenic human BDNF-BAC construct, resembling the expression of endogenous BDNF. Furthermore, BDNF-EGFP mRNA was induced upon treatment with kainic acid in a promotor-specific manner, similarly to that of the endogenous mouse BDNF mRNA. CONCLUSION: Genomic region covering 67 kb of human BDNF gene, 84 kb of upstream and 17 kb of downstream sequences is sufficient to drive tissue-specific and kainic acid-induced expression of the reporter gene in transgenic mice. The pattern of expression of the transgene is highly similar to BDNF gene expression in mouse and human. This is the first study to show that human BDNF gene is regulated by neural activity.