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1.
BMC Neurosci ; 15: 75, 2014 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-24943717

RESUMO

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.


Assuntos
Bioensaio/métodos , Fator Neurotrófico Derivado do Encéfalo/genética , Cromossomos Artificiais Bacterianos/genética , Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Células HeLa , Humanos
2.
Genesis ; 48(4): 214-9, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20186743

RESUMO

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.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Cromossomos Artificiais Bacterianos/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/fisiologia , Transgenes , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Córtex Cerebral/metabolismo , Cromossomos Artificiais Bacterianos/genética , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/metabolismo , Ácido Caínico/farmacologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Ratos
3.
BMC Genomics ; 10: 420, 2009 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-19737418

RESUMO

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.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Análise por Conglomerados , Regulação da Expressão Gênica , Humanos , Camundongos , Ratos , Elementos Reguladores de Transcrição
4.
BMC Neurosci ; 10: 68, 2009 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-19555478

RESUMO

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.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Encéfalo/citologia , Regulação da Expressão Gênica/genética , Neurônios/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cromossomos Artificiais Bacterianos , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Ácido Caínico/farmacologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo
5.
Nat Cell Biol ; 19(5): 421-432, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28368372

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Inativação Gênica , Impressão Genômica , Proteínas Repressoras/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas Cromossômicas não Histona/genética , Técnicas de Cocultura , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Fibroblastos/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Histonas/metabolismo , Fígado/enzimologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteína Nuclear Ligada ao X , Coesinas
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