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1.
Nucleic Acids Res ; 47(4): 1671-1691, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30566651

RESUMO

Fission yeast, Schizosaccharomyces pombe, is an attractive model organism for transcriptional and chromatin biology research. Such research is contingent on accurate annotation of transcription start sites (TSSs). However, comprehensive genome-wide maps of TSSs and their usage across commonly applied laboratory conditions and treatments for S. pombe are lacking. To this end, we profiled TSS activity genome-wide in S. pombe cultures exposed to heat shock, nitrogen starvation, hydrogen peroxide and two commonly applied media, YES and EMM2, using Cap Analysis of Gene Expression (CAGE). CAGE-based annotation of TSSs is substantially more accurate than existing PomBase annotation; on average, CAGE TSSs fall 50-75 bp downstream of PomBase TSSs and co-localize with nucleosome boundaries. In contrast to higher eukaryotes, dispersed TSS distributions are not common in S. pombe. Our data recapitulate known S. pombe stress expression response patterns and identify stress- and media-responsive alternative TSSs. Notably, alteration of growth medium induces changes of similar magnitude as some stressors. We show a link between nucleosome occupancy and genetic variation, and that the proximal promoter region is genetically diverse between S. pombe strains. Our detailed TSS map constitutes a central resource for S. pombe gene regulation research.


Assuntos
Schizosaccharomyces/genética , Estresse Fisiológico/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica , Cromatina/genética , Mapeamento Cromossômico , Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico/efeitos dos fármacos , Genoma Fúngico/genética , Peróxido de Hidrogênio/farmacologia , Nitrogênio/metabolismo , Nucleossomos/genética , Regiões Promotoras Genéticas , Inanição/genética , Estresse Fisiológico/efeitos dos fármacos
2.
Nature ; 507(7493): 455-461, 2014 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-24670763

RESUMO

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.


Assuntos
Atlas como Assunto , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Anotação de Sequência Molecular , Especificidade de Órgãos , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , Predisposição Genética para Doença/genética , Células HeLa , Humanos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética
3.
EMBO Rep ; 17(5): 753-68, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26902262

RESUMO

Retrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we describe a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements. This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome.


Assuntos
Regulação da Expressão Gênica , Retroelementos , Sítio de Iniciação de Transcrição , Sequência de Bases , Catálise , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Modelos Biológicos , Mutação , Nucleossomos , Fenótipo , Estresse Fisiológico , Sequências Repetidas Terminais , Ativação Transcricional
4.
Am J Physiol Gastrointest Liver Physiol ; 302(3): G277-86, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22094602

RESUMO

Upon developmental or environmental cues, the composition of transcription factors in a transcriptional regulatory network is deeply implicated in controlling the signature of the gene expression and thereby specifies the cell or tissue type. Novel methods including ChIP-chip and ChIP-Seq have been applied to analyze known transcription factors and their interacting regulatory DNA elements in the intestine. The intestine is an example of a dynamic tissue where stem cells in the crypt proliferate and undergo a differentiation process toward the villus. During this differentiation process, specific regulatory networks of transcription factors are activated to target specific genes, which determine the intestinal cell fate. The expanding genomewide mapping of transcription factor binding sites and construction of transcriptional regulatory networks provide new insight into how intestinal differentiation occurs. This review summarizes the current overview of the transcriptional regulatory networks driving epithelial differentiation in adult intestine. The novel technologies that have been implied to study these networks are presented and their prospects for implications in future research are also addressed.


Assuntos
Redes Reguladoras de Genes/fisiologia , Mucosa Intestinal/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Imunoprecipitação da Cromatina/métodos , Humanos , Mucosa Intestinal/citologia , Especificidade de Órgãos
5.
J Biol Chem ; 285(33): 25115-25, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20551321

RESUMO

The CDX2 transcription factor is known to play a crucial role in inhibiting proliferation, promoting differentiation and the expression of intestinal specific genes in intestinal cells. The overall effect of CDX2 in intestinal cells has previously been investigated in conditional knock-out mice, revealing a critical role of CDX2 in the formation of the normal intestinal identity. The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. The ChIP-seq technique combines chromatin immunoprecipitation (ChIP) with next generation sequencing resulting in a high throughput experimental method of identifying direct targets of specific transcription factors. The method was applied to CDX2, leading to the identification of the direct binding of CDX2 to several known and novel target genes in the intestinal cell. Examination of the transcript levels of selected genes verified the regulatory role of CDX2 binding. The results place CDX2 as a key node in a transcription factor network controlling the proliferation and differentiation of intestinal cells.


Assuntos
Células Epiteliais/metabolismo , Genoma Humano/genética , Proteínas de Homeodomínio/metabolismo , Intestinos/citologia , Transativadores/metabolismo , Fator de Transcrição CDX2 , Células CACO-2 , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Imunoprecipitação da Cromatina , Biologia Computacional , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/genética , Células Epiteliais/citologia , Fator 4 Nuclear de Hepatócito/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética
6.
J Cell Biochem ; 109(6): 1118-28, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20127720

RESUMO

An important aspect of the cellular differentiation in the intestine is the migration of epithelial cells from the crypt to the villus tip. As homeodomaine transcription factor CDX2 has been suggested to influence cell migration, we performed a genome-wide promoter analysis for CDX2 binding in the differentiated human intestinal cancer cell line Caco-2 in order to identify CDX2-regulated genes involved in cellular migration. The engulfment and cell motility 3 (ELMO3) gene was identified as a potential CDX2 target gene. ELMO3 is an essential upstream regulator of the GTP-binding protein RAC during cell migration. However, no information is available about the transcriptional regulation of the ELMO3 gene. The aim of this study was to investigate the potential role of CDX2 in the regulation of the ELMO3 promoter activity. Electrophoretic mobility shift assays showed that CDX2 bound to conserved CDX2 sequences and mutations of the CDX2-binding sites, significantly reduced the promoter activity. Reporter gene assays demonstrated that the region mediating ELMO3 basal transcriptional activity to be located between -270 and -31 bp. Sequence analysis revealed no typical TATA-box, but four GC-rich sequences. In vitro analyses (electrophoretic mobility shift assays and promoter analyses) demonstrate that the SP1-binding sites are likely to play an important role in regulating the ELMO3 promoter activity. Furthermore, we showed here that CDX2 and SP1 can activate the ELMO3 promoter. Taken together, the present study reports the first characterization of the ELMO3 promoter and suggests a significant role of CDX2 in the basal transcriptional regulation of the intestine-specific expression of ELMO3, possibly through interaction with SP1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/metabolismo , Transativadores/metabolismo , Fator de Transcrição CDX2 , Células CACO-2 , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Células HT29 , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/genética , Transativadores/genética
7.
BMC Gastroenterol ; 9: 68, 2009 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-19761587

RESUMO

BACKGROUND: The role of HNF4alpha has been extensively studied in hepatocytes and pancreatic beta-cells, and HNF4alpha is also regarded as a key regulator of intestinal epithelial cell differentiation. The aim of the present work is to identify novel HNF4alpha target genes in the human intestinal epithelial cells in order to elucidate the role of HNF4alpha in the intestinal differentiation progress. METHODS: We have performed a ChIP-chip analysis of the human intestinal cell line Caco-2 in order to make a genome-wide identification of HNF4alpha binding to promoter regions. The HNF4alpha ChIP-chip data was matched with gene expression and histone H3 acetylation status of the promoters in order to identify HNF4alpha binding to actively transcribed genes with an open chromatin structure. RESULTS: 1,541 genes were identified as potential HNF4alpha targets, many of which have not previously been described as being regulated by HNF4alpha. The 1,541 genes contributed significantly to gene ontology (GO) pathways categorized by lipid and amino acid transport and metabolism. An analysis of the homeodomain transcription factor Cdx-2 (CDX2), the disaccharidase trehalase (TREH), and the tight junction protein cingulin (CGN) promoters verified that these genes are bound by HNF4alpha in Caco2 cells. For the Cdx-2 and trehalase promoters the HNF4alpha binding was verified in mouse small intestine epithelium. CONCLUSION: The HNF4alpha regulation of the Cdx-2 promoter unravels a transcription factor network also including HNF1alpha, all of which are transcription factors involved in intestinal development and gene expression.


Assuntos
Mapeamento Cromossômico , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Fator de Transcrição CDX2 , Células CACO-2 , Diferenciação Celular , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Trealase/metabolismo
8.
FEBS Lett ; 592(4): 631-643, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29355922

RESUMO

Targeting of ArabidopsisPHABULOSA (PHB) mRNA by miR166 has been implicated in gene body methylation at the PHB locus. We report that the PHB locus produces an array of stable nuclear RNA species that are neither polyadenylated nor capped. Their biogenesis requires neither RNA polymerases IV/V nor miR166-guided cleavage. The PHB RNAs are insensitive to mutation of nuclear RNA decay pathways and are conserved in several Brassicaceae species, suggesting functional relevance. Similar RNA species are also produced by another body-methylated locus encoding the miR414 target eIF2. Our data reveal the existence of a new class of genic nuclear RNA species.


Assuntos
Arabidopsis/genética , RNA Nuclear/genética , RNA de Plantas/genética , Arabidopsis/citologia , Arabidopsis/metabolismo , Metilação de DNA , Fator de Iniciação 2 em Eucariotos/metabolismo , Exossomos/genética , Loci Gênicos/genética , MicroRNAs/genética , Mutação , Clivagem do RNA , RNA Mensageiro/genética , RNA Nuclear/metabolismo , RNA de Plantas/metabolismo , Especificidade da Espécie
9.
Nat Commun ; 9(1): 1661, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29695774

RESUMO

Inflammatory bowel disease (IBD) is a chronic intestinal disorder, with two main types: Crohn's disease (CD) and ulcerative colitis (UC), whose molecular pathology is not well understood. The majority of IBD-associated SNPs are located in non-coding regions and are hard to characterize since regulatory regions in IBD are not known. Here we profile transcription start sites (TSSs) and enhancers in the descending colon of 94 IBD patients and controls. IBD-upregulated promoters and enhancers are highly enriched for IBD-associated SNPs and are bound by the same transcription factors. IBD-specific TSSs are associated to genes with roles in both inflammatory cascades and gut epithelia while TSSs distinguishing UC and CD are associated to gut epithelia functions. We find that as few as 35 TSSs can distinguish active CD, UC, and controls with 85% accuracy in an independent cohort. Our data constitute a foundation for understanding the molecular pathology, gene regulation, and genetics of IBD.


Assuntos
Colite Ulcerativa/genética , Doença de Crohn/genética , Sequências Reguladoras de Ácido Nucleico/genética , Adulto , Biópsia , Estudos de Casos e Controles , Estudos de Coortes , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/patologia , Colo/diagnóstico por imagem , Colo/patologia , Colonoscopia , Doença de Crohn/diagnóstico , Doença de Crohn/patologia , Feminino , Humanos , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA , Regulação para Cima
10.
NPJ Genom Med ; 2: 3, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29263823

RESUMO

Crohn's disease is associated with an altered innate immune response of pathogenic importance. This altered response can be associated to loss-of-function polymorphisms in the NOD2 (nucleotide-binding oligomerization domain-containing protein 2) gene, but also changes in transcriptional and post-transcriptional regulatory layers, including microRNA activity. Here, we characterized the link between NOD2 genotype and inflammatory-mediated changes in innate signaling by studying transcriptional and post-transcriptional activity in response to NOD2-agonist muramyl dipeptide in monocytes from healthy controls, and Crohn's disease patients with and without NOD2 loss-of-function polymorphisms. We measured the expression of genes and microRNAs in monocytes from these subjects after stimulation with muramyl dipeptide. Gene expression profiles mainly distinguished the actual muramyl dipeptide response, but not the genotype. A hyper-responsive phenotype was found in Crohn's disease patients without NOD2 mutations, characterized by upregulated cytokine receptors and general downregulation of microRNA expression. Conversely, microRNA expression could identify genotype-specific differences between subject groups but exhibited little change upon muramyl dipeptide treatment. Only two microRNAs showed muramyl dipeptide-induced response, including miR-155, which was found to regulate multiple genes and whose host gene was one of the highest muramyl dipeptide responders. miR-155 was upregulated in Crohn's disease patients with NOD2 mutations following lipopolysaccharide and Escherichia coli treatment, but the upregulation was substantially reduced upon muramyl dipeptide treatment. While Crohn's disease patients with NOD2 mutations on average showed a reduced muramyl dipeptide response, the cohort exhibited large individual variance: a small subset had inflammatory responses almost comparable to wild-type patients on both gene and miR-155 regulatory levels.

11.
ACS Nano ; 11(4): 3597-3613, 2017 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-28345861

RESUMO

Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube (MWCNT) Mitsui-7. Our results revealed a massive gene regulatory response, where expression of key inflammatory genes (e.g., Csf3, Il24, and Fgf23) was increased >100-fold 24 h after Mitsui-7 exposure. Many of the Mitsui-7-responsive transcription start sites were alternative transcription start sites for known genes, and the number of alternative transcription start sites used in a given gene was correlated with overall Mitsui-7 response. Strikingly, genes that were up-regulated after Mitsui-7 exposure only through their main annotated transcription start site were linked to inflammatory and defense responses, while genes up-regulated only through alternative transcription start sites were functionally heterogeneous and not inflammation-associated. Furthermore, we identified almost 12 000 active enhancers, many of which were Mitsui-7-responsive, and we identified similarly responding putative target genes. Overall, our study provides the location and activity of Mitsui-7-induced enhancers and transcription start sites, providing a useful resource for targeted experiments elucidating the biological effects of nanomaterials and the identification of biomarkers for early detection of MWCNT-induced inflammation.


Assuntos
Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Animais , Fator de Crescimento de Fibroblastos 23 , Inflamação/genética , Injeção Intratimpânica , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nanotubos de Carbono/química , Sítio de Iniciação de Transcrição/efeitos dos fármacos
12.
Cell Rep ; 16(9): 2317-26, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27545890

RESUMO

Metabolically healthy obese subjects display preserved insulin sensitivity and a beneficial white adipose tissue gene expression pattern. However, this observation stems from fasting studies when insulin levels are low. We investigated adipose gene expression by 5'Cap-mRNA sequencing in 17 healthy non-obese (NO), 21 insulin-sensitive severely obese (ISO), and 30 insulin-resistant severely obese (IRO) subjects, before and 2 hr into a hyperinsulinemic euglycemic clamp. ISO and IRO subjects displayed a clear but globally similar transcriptional response to insulin, which differed from the small effects observed in NO subjects. In the obese, 231 genes were altered; 71 were enriched in ISO subjects (e.g., phosphorylation processes), and 52 were enriched in IRO subjects (e.g., cellular stimuli). Common cardio-metabolic risk factors and gender do not influence these findings. This study demonstrates that differences in the acute transcriptional response to insulin are primarily driven by obesity per se, challenging the notion of healthy obese adipose tissue, at least in severe obesity.


Assuntos
Tecido Adiposo/efeitos dos fármacos , Resistência à Insulina/genética , Insulina/administração & dosagem , Obesidade/genética , Transcrição Gênica , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Adulto , Glicemia/metabolismo , Pressão Sanguínea , Estudos de Casos e Controles , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Jejum , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Técnica Clamp de Glucose , Humanos , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Obesidade/metabolismo , Obesidade/patologia , Índice de Gravidade de Doença , Triglicerídeos/sangue
13.
Artigo em Inglês | MEDLINE | ID: mdl-28025337

RESUMO

Genomics consortia have produced large datasets profiling the expression of genes, micro-RNAs, enhancers and more across human tissues or cells. There is a need for intuitive tools to select subsets of such data that is the most relevant for specific studies. To this end, we present SlideBase, a web tool which offers a new way of selecting genes, promoters, enhancers and microRNAs that are preferentially expressed/used in a specified set of cells/tissues, based on the use of interactive sliders. With the help of sliders, SlideBase enables users to define custom expression thresholds for individual cell types/tissues, producing sets of genes, enhancers etc. which satisfy these constraints. Changes in slider settings result in simultaneous changes in the selected sets, updated in real time. SlideBase is linked to major databases from genomics consortia, including FANTOM, GTEx, The Human Protein Atlas and BioGPS.Database URL: http://slidebase.binf.ku.dk.


Assuntos
Bases de Dados Genéticas , Elementos Facilitadores Genéticos , Genoma Humano , MicroRNAs/genética , Proteínas/genética , Análise de Sequência de DNA/métodos , Projeto Genoma Humano , Humanos
14.
Nat Commun ; 5: 5336, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25387874

RESUMO

Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.


Assuntos
Estabilidade de RNA/fisiologia , RNA/classificação , Exossomos/genética , Exossomos/fisiologia , Células HeLa , Humanos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , RNA/fisiologia , Estabilidade de RNA/genética , RNA não Traduzido/fisiologia , Iniciação da Transcrição Genética/fisiologia , Transcrição Gênica/fisiologia
15.
DNA Res ; 21(6): 569-83, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24990076

RESUMO

The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers.


Assuntos
Mapeamento Cromossômico , Elementos de Resposta/fisiologia , Iniciação da Transcrição Genética/efeitos dos fármacos , Iniciação da Transcrição Genética/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Células CACO-2 , Estudo de Associação Genômica Ampla , Humanos , Laminina/biossíntese , Laminina/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
16.
Methods Mol Biol ; 1038: 213-31, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23872978

RESUMO

Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR amplification, Illumina adapters and index sequences are introduced, thereby allowing amplicons to be pooled and sequenced on the standard Illumina platform for genomic DNA sequencing. Moreover, we demonstrate how to map sequencing reads and perform analysis of the sequencing data with freely available tools that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Polimerase Dirigida por RNA/análise , Transcrição Reversa , Animais , Bacteriófagos/enzimologia , DNA Complementar/análise , DNA Complementar/metabolismo , Camundongos , Reação em Cadeia da Polimerase/métodos , RNA Ligase (ATP)/análise , RNA Ligase (ATP)/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Análise de Sequência de DNA/métodos
17.
Nat Struct Mol Biol ; 20(8): 923-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23851456

RESUMO

Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3' ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.


Assuntos
Poliadenilação/fisiologia , Regiões Promotoras Genéticas/genética , Estabilidade de RNA/fisiologia , Transcrição Gênica/fisiologia , Sequência de Bases , Northern Blotting , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Poliadenilação/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA/genética , Sítio de Iniciação de Transcrição/fisiologia , Transcrição Gênica/genética
18.
Eur J Hum Genet ; 18(6): 733-6, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20068592

RESUMO

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T>G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.


Assuntos
Proteínas de Membrana/genética , Polidactilia/genética , Elementos Reguladores de Transcrição/genética , Polegar/anormalidades , Fatores de Transcrição/metabolismo , Sequência de Bases , Células CACO-2 , Família , Humanos , Proteínas de Membrana/metabolismo , Linhagem , Mutação Puntual/fisiologia , Polidactilia/complicações , Polidactilia/metabolismo , Ligação Proteica , Especificidade por Substrato
19.
Brain Res Bull ; 77(1): 1-7, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18639740

RESUMO

Systemically administered human recombinant erythropoietin (EPO) may have the potential to reduce the cognitive and behavioural symptoms of mechanical brain injury. In a series of studies we address this possibility. Previously, we studied the effects of EPO given to fimbria-fornix transected rats at the moment of injury. We have found that such treatment improves substantially the posttraumatic acquisition of allocentric place learning tasks administered in a water maze and in an 8-arm radial maze as well as a spatial delayed alternation task administered in a T-maze. It is, however, essential also to evaluate this clinically important ability of EPO after other types of mechanical brain injury. Consequently, we presently studied the effects of similarly administered EPO in rats subjected to bilateral subpial aspiration of the anteromedial prefrontal cortex as well as control operated rats, respectively. We evaluated the posttraumatic behavioural/cognitive abilities of these animals in a spatial delayed alternation task performed in a T-maze. Administration of EPO to the prefrontally ablated rats was associated with a reduction of the lesion-associated behavioural impairment--while such an impairment was clearly seen in the saline injected prefrontally ablated group. In sham operated rats administration of EPO did not influence the task acquisition significantly. The results of the present study confirm our previous demonstrations that EPO is able to reduce the behavioural/cognitive consequences of mechanical brain injury. This ability is emphasized by its relative independence on the type of lesion as well as the neural structure affected.


Assuntos
Cognição/efeitos dos fármacos , Eritropoetina/farmacologia , Aprendizagem em Labirinto/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Percepção Espacial/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Cognição/fisiologia , Eritropoetina/administração & dosagem , Injeções Intraperitoneais , Masculino , Aprendizagem em Labirinto/fisiologia , Córtex Pré-Frontal/lesões , Córtex Pré-Frontal/patologia , Ratos , Ratos Wistar , Proteínas Recombinantes , Percepção Espacial/fisiologia
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