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1.
Biochem Cell Biol ; 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31479623

RESUMO

We previously demonstrated that genome reorganization, through chromosome territory repositioning, occurred concurrently with significant changes in gene expression in normal primary human fibroblasts treated with the drug rapamycin, or stimulated into quiescence. Although these events occurred concomitantly, it is unclear how specific changes in gene expression relate to reorganization of the genome at higher resolution. Using computational analyses, genome organization assays and microscopy, the relationship between chromosome territory positioning and gene expression was investigated. We determined that despite relocation of chromosome territories, there was no substantial bias in the proportion of genes changing expression on any one chromosome, including chromosomes 10 and 18. Computational analyses identified that clusters of serum deprivation and rapamycin-responsive genes along the linear extent of chromosomes. Chromosome conformation capture (3C) analysis demonstrated the strengthening or loss of specific long-range chromatin interactions in response to rapamycin and quiescence induction, including a cluster of genes containing Interleukin-8 and several chemokine genes on chromosome 4. We further observed that the LIF gene, which is highly induced upon rapamycin treatment, strengthened interactions with up- and down-stream intergenic regions. Our findings indicate that the re-positioning of chromosome territories in response to cell stimuli, this does not reflect gene expression changes occurring within physically clustered groups of genes.

2.
Genes Dev ; 33(15-16): 1069-1082, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31221664

RESUMO

Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of <2 h. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2, and STAT3) that together facilitate association of KLF4 with RNA polymerase II. In addition, the KLF4 DNA-binding and transactivation domains are required for optimal KLF4 protein stability. Posttranslational modification of KLF4 destabilizes the protein as cells exit the pluripotent state, and mutations that prevent this destabilization also prevent differentiation. These data indicate that the core pluripotency transcription factors are integrated by posttranslational mechanisms to maintain the pluripotent state and identify mutations that increase KLF4 protein stability while maintaining transcription factor function.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Células-Tronco Embrionárias , Células HEK293 , Humanos , Camundongos , Mutação/genética , Domínios Proteicos , Estabilidade Proteica , Proteólise , RNA Polimerase II/metabolismo , Transdução de Sinais , Ubiquitinação
3.
Cell Stem Cell ; 24(5): 681-683, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31051130

RESUMO

Exit from the naive pluripotent state occurs through a series of changes in the gene regulatory circuitry, allowing cells to become primed for lineage commitment. In this issue of Cell Stem Cell, Kalkan et al. (2019) show that three transcription regulators are required for naive mouse embryonic stem cells (ESCs) to exit the pluripotent state.

4.
Bioinformatics ; 35(18): 3232-3239, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30753279

RESUMO

MOTIVATION: Mammalian genomes can contain thousands of enhancers but only a subset are actively driving gene expression in a given cellular context. Integrated genomic datasets can be harnessed to predict active enhancers. One challenge in integration of large genomic datasets is the increasing heterogeneity: continuous, binary and discrete features may all be relevant. Coupled with the typically small numbers of training examples, semi-supervised approaches for heterogeneous data are needed; however, current enhancer prediction methods are not designed to handle heterogeneous data in the semi-supervised paradigm. RESULTS: We implemented a Dirichlet Process Heterogeneous Mixture model that infers Gaussian, Bernoulli and Poisson distributions over features. We derived a novel variational inference algorithm to handle semi-supervised learning tasks where certain observations are forced to cluster together. We applied this model to enhancer candidates in mouse heart tissues based on heterogeneous features. We constrained a small number of known active enhancers to appear in the same cluster, and 47 additional regions clustered with them. Many of these are located near heart-specific genes. The model also predicted 1176 active promoters, suggesting that it can discover new enhancers and promoters. AVAILABILITY AND IMPLEMENTATION: We created the 'dphmix' Python package: https://pypi.org/project/dphmix/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

5.
Stem Cell Reports ; 10(4): 1308-1323, 2018 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-29526737

RESUMO

Cooperative action of a transcription factor complex containing OCT4, SOX2, NANOG, and KLF4 maintains the naive pluripotent state; however, less is known about the mechanisms that disrupt this complex, initiating exit from pluripotency. We show that, as embryonic stem cells (ESCs) exit pluripotency, KLF4 protein is exported from the nucleus causing rapid decline in Nanog and Klf4 transcription; as a result, KLF4 is the first pluripotency transcription factor removed from transcription-associated complexes during differentiation. KLF4 nuclear export requires ERK activation, and phosphorylation of KLF4 by ERK initiates interaction of KLF4 with nuclear export factor XPO1, leading to KLF4 export. Mutation of the ERK phosphorylation site in KLF4 (S132) blocks KLF4 nuclear export, the decline in Nanog, Klf4, and Sox2 mRNA, and differentiation. These findings demonstrate that relocalization of KLF4 to the cytoplasm is a critical first step in exit from the naive pluripotent state and initiation of ESC differentiation.

6.
Fam Syst Health ; 35(4): 450-462, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29283612

RESUMO

INTRODUCTION: Many military service members with PTSD do not receive evidence-based specialty behavioral health treatment because of perceived barriers and stigma. Behavioral health providers in primary care can deliver brief, effective treatments expanding access and reducing barriers and stigma. The purpose of this randomized clinical trial was to determine if a brief cognitive-behavior therapy delivered in primary care using the Primary Care Behavioral Health model would be effective at reducing PTSD and co-occurring symptoms. METHOD: A total of 67 service members (50 men, 17 women) were randomized to receive a brief, trauma-focused intervention developed for the primary care setting called Prolonged Exposure for Primary Care (PE-PC) or a delayed treatment minimal contact control condition. Inclusion criteria were significant PTSD symptoms following military deployment, medication stability, and interest in receiving treatment for PTSD symptoms in primary care. Exclusion criteria were moderate or greater risk of suicide, severe brain injury, or alcohol/substance use at a level that required immediate treatment. Assessments were completed at baseline, posttreatment/postminimal contact control, and at 8-week and 6-month posttreatment follow-up points. Primary measures were the PTSD Symptom Scale-Interview and the PTSD Checklist-Stressor-Specific. RESULTS: PE-PC resulted in larger reduction in PTSD severity and general distress than the minimal contact control. Delayed treatment evidenced medium to large effects comparable to the immediate intervention group. Treatment benefits persisted through the 6-month follow-up of the study. DISCUSSION: PE-PC delivered in integrated primary care is effective for the treatment of PTSD and co-occurring symptoms and may help reduce barriers and stigma found in specialty care settings. (PsycINFO Database Record


Assuntos
Militares/estatística & dados numéricos , Atenção Primária à Saúde/métodos , Transtornos de Estresse Pós-Traumáticos/terapia , Adulto , Campanha Afegã de 2001- , Terapia Cognitivo-Comportamental/métodos , Terapia Cognitivo-Comportamental/normas , Feminino , Humanos , Guerra do Iraque 2003-2011 , Masculino , Pessoa de Meia-Idade , Atenção Primária à Saúde/normas , Psicometria/instrumentação , Psicometria/métodos , Resultado do Tratamento
7.
Genome Res ; 27(2): 246-258, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27895109

RESUMO

Transcriptional enhancers are critical for maintaining cell-type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.


Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Embrionárias Murinas/metabolismo , Transcrição Genética , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Deleção de Genes , Camundongos
8.
J Biomed Nanotechnol ; 13(2): 221-31, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29377653

RESUMO

Biomedical applications for metal and metal oxide nanoparticles are rapidly increasing. Here their functional impact on two well-characterized model enzymes, Luciferase (Luc) or ß-galactosidase (ß-Gal) was quantitatively compared. Nickel oxide nanoparticle (NiO-NP) activated ß-Gal (>400% control) and boron carbide nanoparticle (B4C-NP) inhibited Luc(<10% control), whereas zinc oxide (ZnO-NP) and cobalt oxide (Co3O4-NP) activated ß-Gal to a lesser extent and magnesium oxide (MgO) moderately inhibited both enzymes. Melanoma specific killing was in the order; ZnO > B4C ≥ Cu > MgO > Co3O4 > Fe2O3 > NiO, ZnO-NP inhibiting B16F10 and A375 cells as well as ERK enzyme (>90%) and several other cancer-associated kinases (AKT, CREB, p70S6K). ZnO-NP or nanobelt (NB) serve as photoluminescence (PL) cell labels and inhibit 3-D multi-cellular tumor spheroid (MCTS) growth and were tested in a mouse melanoma model. These results demonstrate nanoparticle and enzyme specific biochemical activity and suggest their utility as new tools to explore the important model metastatic foci 3-D environment and their chemotherapeutic potential.


Assuntos
Antineoplásicos/farmacologia , Melanoma Experimental/metabolismo , Nanopartículas Metálicas/química , Esferoides Celulares/efeitos dos fármacos , Óxido de Zinco/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Luciferases/análise , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Metais Pesados/farmacologia , Camundongos , Óxido de Zinco/química , beta-Galactosidase/análise , beta-Galactosidase/efeitos dos fármacos , beta-Galactosidase/metabolismo
9.
J Vis Exp ; (110): e53552, 2016 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-27078492

RESUMO

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus(129) x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , Elementos Facilitadores Genéticos/genética , Recombinação Homóloga/genética , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Deleção de Genes , Expressão Gênica/fisiologia , Técnicas de Genotipagem , Camundongos , Plasmídeos , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Sequências Reguladoras de Ácido Nucleico/genética , Transfecção
10.
Methods Mol Biol ; 1402: 63-71, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26721484

RESUMO

Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression. Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long noncoding RNAs. Here, we describe nuclear-RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long noncoding RNAs.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA Nuclear/genética , RNA Nuclear/isolamento & purificação , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Fracionamento Celular/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Poliadenilação , RNA Longo não Codificante/genética , RNA Longo não Codificante/isolamento & purificação
11.
Nucleus ; 6(6): 490-506, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26652669

RESUMO

Rapamycin is a well-known inhibitor of the Target of Rapamycin (TOR) signaling cascade; however, the impact of this drug on global genome function and organization in normal primary cells is poorly understood. To explore this impact, we treated primary human foreskin fibroblasts with rapamycin and observed a decrease in cell proliferation without causing cell death. Upon rapamycin treatment chromosomes 18 and 10 were repositioned to a location similar to that of fibroblasts induced into quiescence by serum reduction. Although similar changes in positioning occurred, comparative transcriptome analyses demonstrated significant divergence in gene expression patterns between rapamycin-treated and quiescence-induced fibroblasts. Rapamycin treatment induced the upregulation of cytokine genes, including those from the Interleukin (IL)-6 signaling network, such as IL-8 and the Leukemia Inhibitory Factor (LIF), while quiescent fibroblasts demonstrated up-regulation of genes involved in the complement and coagulation cascade. In addition, genes significantly up-regulated by rapamycin treatment demonstrated increased promoter occupancy of the transcription factor Signal Transducer and Activator of Transcription 5A/B (STAT5A/B). In summary, we demonstrated that the treatment of fibroblasts with rapamycin decreased proliferation, caused chromosome territory repositioning and induced STAT5A/B-mediated changes in gene expression enriched for cytokines.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Sirolimo/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Actinas/metabolismo , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Fator Inibidor de Leucemia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT5/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Proteínas Supressoras de Tumor/genética , Regulação para Cima/efeitos dos fármacos
12.
R Soc Open Sci ; 2(9): 150402, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26473061

RESUMO

DNA microarrays and RNA sequencing (RNA-seq) are major technologies for performing high-throughput analysis of transcript abundance. Recently, concerns have been raised regarding the concordance of data derived from the two techniques. Using cDNA libraries derived from normal human foreskin fibroblasts, we measured changes in transcript abundance as cells transitioned from proliferative growth to quiescence using both DNA microarrays and RNA-seq. The internal reproducibility of the RNA-seq data was greater than that of the microarray data. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarray values were moderate. The two technologies had good agreement when considering probes with the largest (both positive and negative) fold change (FC) values. An independent technique, quantitative reverse-transcription PCR (qRT-PCR), was used to measure the FC of 76 genes between proliferative and quiescent samples, and a higher correlation was observed between the qRT-PCR data and the RNA-seq data than between the qRT-PCR data and the microarray data.

13.
Genes (Basel) ; 6(3): 641-61, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26193323

RESUMO

Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells.

14.
Genome Res ; 25(4): 582-97, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25752748

RESUMO

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.


Assuntos
Sítios de Ligação/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regiões Promotoras Genéticas/genética , Animais , Cromatina/genética , Células-Tronco Embrionárias/citologia , Epigênese Genética , Histonas/genética , Fígado/citologia , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Genes Dev ; 28(24): 2699-711, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25512558

RESUMO

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.


Assuntos
Diferenciação Celular , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Células Cultivadas , Camundongos , Família Multigênica/genética , Placa Neural/citologia , Regiões Promotoras Genéticas/genética , Deleção de Sequência/genética
16.
Biochem Cell Biol ; 91(1): 22-30, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23442138

RESUMO

Transcription occurs at distinct nuclear compartments termed transcription factories that are specialized for transcription by 1 of the 3 polymerase complexes (I, II, or III). Protein-coding genes appear to move in and out of RNA polymerase II (RNAPII) compartments as they are expressed and silenced. In addition, transcription factories are sites where several transcription units, either from the same chromosome or different chromosomes, are transcribed. Chromosomes occupy distinct territories in the interphase nucleus with active genes preferentially positioned on the periphery or even looped out of the territory. These chromosome territories have been observed to intermingle in the nucleus, and multiple interactions among different chromosomes have been identified in genome-wide studies. Deep sequencing of the transcriptome and RNAPII associated on DNA obtained by chromatin immunoprecipitation have revealed a plethora of noncoding transcription and intergenic accumulations of RNAPII that must also be considered in models of genome function. The organization of transcription into distinct regions of the nucleus has changed the way we view transcription with the evolving model for silencing or activation of gene expression involving physical relocation of the transcription unit to a silencing or activation compartment, thus, highlighting the need to consider the process of transcription in the 3-dimensional nuclear space.


Assuntos
Núcleo Celular/genética , Cromatina , Genoma , RNA Polimerase II/genética , Transcrição Genética , Animais , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Inativação Gênica , Estudo de Associação Genômica Ampla , Humanos , RNA Polimerase II/metabolismo
17.
PLoS One ; 7(11): e49274, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209567

RESUMO

In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.


Assuntos
Células Eritroides/metabolismo , RNA Nuclear/genética , Transcriptoma , Animais , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sequências Reguladoras de Ácido Nucleico , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transcrição Genética
18.
Nat Protoc ; 7(7): 1335-50, 2012 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-22722369

RESUMO

Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Cromossomos/química , Epigenômica/métodos , Conformação de Ácido Nucleico , Biotinilação , Cromatina/metabolismo , Cromossomos/metabolismo , Primers do DNA/genética
19.
BMC Genomics ; 13: 152, 2012 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-22537144

RESUMO

BACKGROUND: Epigenetic modifications, transcription factor (TF) availability and differences in chromatin folding influence how the genome is interpreted by the transcriptional machinery responsible for gene expression. Enhancers buried in non-coding regions are found to be associated with significant differences in histone marks between different cell types. In contrast, gene promoters show more uniform modifications across cell types. Here we used histone modification and chromatin-associated protein ChIP-Seq data sets in mouse embryonic stem (ES) cells as well as genomic features to identify functional enhancer regions. Using co-bound sites of OCT4, SOX2 and NANOG (co-OSN, validated enhancers) and co-bound sites of MYC and MYCN (limited enhancer activity) as enhancer positive and negative training sets, we performed multinomial logistic regression with LASSO regularization to identify key features. RESULTS: Cross validations reveal that a combination of p300, H3K4me1, MED12 and NIPBL features to be top signatures of co-OSN regions. Using a model from 10 signatures, 83% of top 1277 putative 1 kb enhancer regions (probability greater than or equal to 0.8) overlapped with at least one TF peak from 7 mouse ES cell ChIP-Seq data sets. These putative enhancers are associated with increased gene expression of neighbouring genes and significantly enriched in multiple TF bound loci in agreement with combinatorial models of TF binding. Furthermore, we identified several motifs of known TFs significantly enriched in putative enhancer regions compared to random promoter regions and background. Comparison with an active H3K27ac mark in various cell types confirmed cell type-specificity of these enhancers. CONCLUSIONS: The top enhancer signatures we identified (p300, H3K4me1, MED12 and NIPBL) will allow for the identification of cell type-specific enhancer regions in diverse cell types.


Assuntos
Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Genoma , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Camundongos , Modelos Genéticos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
PLoS One ; 7(12): e52880, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23285212

RESUMO

The Lim domain only 2 (Lmo2) gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Several distal regulatory elements have been identified upstream of the Lmo2 gene in the human and mouse genomes that are capable of enhancing reporter gene expression in erythroid cells and may be responsible for the high level transcription of Lmo2 in the erythroid lineage. In this study we investigate how these elements regulate transcription of Lmo2 and whether or not they function cooperatively in the endogenous context. Chromosome conformation capture (3C) experiments show that chromatin-chromatin interactions exist between upstream regulatory elements and the Lmo2 promoter in erythroid cells but that these interactions are absent from kidney where Lmo2 is transcribed at twelve fold lower levels. Specifically, long range chromatin-chromatin interactions occur between the Lmo2 proximal promoter and two broad regions, 3-31 and 66-105 kb upstream of Lmo2, which we term the proximal and distal control regions for Lmo2 (pCR and dCR respectively). Each of these regions is bound by several transcription factors suggesting that multiple regulatory elements cooperate in regulating high level transcription of Lmo2 in erythroid cells. Binding of CTCF and cohesin which support chromatin loops at other loci were also found within the dCR and at the Lmo2 proximal promoter. Intergenic transcription occurs throughout the dCR in erythroid cells but not in kidney suggesting a role for these intergenic transcripts in regulating Lmo2, similar to the broad domain of intergenic transcription observed at the human ß-globin locus control region. Our data supports a model in which the dCR functions through a chromatin looping mechanism to contact and enhance Lmo2 transcription specifically in erythroid cells. Furthermore, these chromatin loops are supported by the cohesin complex recruited to both CTCF and transcription factor bound regions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Eritroides/metabolismo , Proteínas com Domínio LIM/genética , Regiões Promotoras Genéticas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Humanos , Proteínas com Domínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Conformação de Ácido Nucleico , Especificidade de Órgãos/genética , Transcrição Genética , Transfecção
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