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2.
Nat Genet ; 51(12): 1664-1669, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31784727

RESUMO

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases1-4. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types5,6. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Animais , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Desacetilase 6 de Histona/genética , Humanos , Hibridização in Situ Fluorescente , Células K562 , Camundongos , Modelos Genéticos , RNA Guia
3.
Nat Genet ; 51(12): 1723-1731, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31784729

RESUMO

WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via ß-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver.


Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Genes myc , Fatores de Transcrição/genética , Via de Sinalização Wnt/genética , Colo/citologia , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/fisiologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Processamento Pós-Transcricional do RNA , Fatores de Transcrição/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
4.
Nat Genet ; 51(12): 1714-1722, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31784732

RESUMO

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.


Assuntos
Histonas/metabolismo , Rabdomiossarcoma/genética , Fatores de Transcrição/genética , Acetilação , Benzamidas/farmacologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Humanos , Piridinas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA , Fatores de Transcrição SOXE/genética , Análise de Célula Única
5.
Mol Biol (Mosk) ; 53(6): 1038-1048, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31876282

RESUMO

Transcriptional enhancers in the cell nuclei typically interact with the target promoters in cis over long stretches of chromatin, but the mechanism of this communication remains unknown. Previously we have developed a defined in vitro system for quantitative analysis of the rate of distant enhancer-promoter communication (EPC) and have shown that the chromatin fibers maintain efficient distant EPC in cis. Here we investigate the roles of linker histone H1 and HMGN5 protein in EPC. A considerable negative effect of histone H1 on EPC depending on its C- and N-tails was shown. Protein HMGN5 that affects chromatin compaction and is associated with active chromatin counteracts EPC inhibition by H1. The data suggest that the efficiency of the interaction between the enhancer and the promoter depends on the structure and dynamics of the chromatin fiber localized between them and can be regulated by proteins associated with chromatin.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Proteínas HMGN/metabolismo , Histonas/metabolismo , Cromatina/química , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética
6.
Nat Genet ; 51(11): 1588-1595, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31676868

RESUMO

The early stages of type 1 diabetes (T1D) are characterized by local autoimmune inflammation and progressive loss of insulin-producing pancreatic ß cells. Here we show that exposure to proinflammatory cytokines reveals a marked plasticity of the ß-cell regulatory landscape. We expand the repertoire of human islet regulatory elements by mapping stimulus-responsive enhancers linked to changes in the ß-cell transcriptome, proteome and three-dimensional chromatin structure. Our data indicate that the ß-cell response to cytokines is mediated by the induction of new regulatory regions as well as the activation of primed regulatory elements prebound by islet-specific transcription factors. We find that T1D-associated loci are enriched with newly mapped cis-regulatory regions and identify T1D-associated variants disrupting cytokine-responsive enhancer activity in human ß cells. Our study illustrates how ß cells respond to a proinflammatory environment and implicate a role for stimulus response islet enhancers in T1D.


Assuntos
Cromatina/genética , Citocinas/farmacologia , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Células Secretoras de Insulina/metabolismo , Transcriptoma , Cromatina/química , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/patologia , Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Fatores de Transcrição
7.
Hum Genet ; 138(11-12): 1301-1311, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686214

RESUMO

Haploinsufficiency of FOXF1 causes alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a lethal neonatal lung developmental disorder. We describe two similar heterozygous CNV deletions involving the FOXF1 enhancer and re-analyze FOXF1 missense mutation, all associated with an unexpectedly mitigated disease phenotype. In one case, the deletion of the maternal allele of the FOXF1 enhancer caused pulmonary hypertension and histopathologically diagnosed MPV without the typical ACD features. In the second case, the deletion of the paternal enhancer resulted in ACDMPV rather than the expected neonatal lethality. In both cases, FOXF1 expression in lung tissue was higher than usually seen or expected in patients with similar deletions, suggesting an increased activity of the remaining allele of the enhancer. Sequencing of these alleles revealed two rare SNVs, rs150502618-A and rs79301423-T, mapping to the partially overlapping binding sites for TFAP2s and CTCF in the core region of the enhancer. Moreover, in a family with three histopathologically-diagnosed ACDMPV siblings whose missense FOXF1 mutation was inherited from the healthy non-mosaic carrier mother, we have identified a rare SNV rs28571077-A within 2-kb of the above-mentioned non-coding SNVs in the FOXF1 enhancer in the mother, that was absent in the affected newborns and 13 unrelated ACDMPV patients with CNV deletions of this genomic region. Based on the low population frequencies of these three variants, their absence in ACDMPV patients, the results of reporter assay, RNAi and EMSA experiments, and in silico predictions, we propose that the described SNVs might have acted on FOXF1 enhancer as hypermorphs.


Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/genética , Mutação de Sentido Incorreto , Síndrome da Persistência do Padrão de Circulação Fetal/prevenção & controle , Polimorfismo de Nucleotídeo Único , Deleção de Sequência , Adulto , Criança , Feminino , Impressão Genômica , Humanos , Recém-Nascido , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Fenótipo , Prognóstico
8.
BMC Bioinformatics ; 20(1): 560, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31703553

RESUMO

BACKGROUND: With the growth of available sequenced datasets, analysis of heterogeneous processed data can answer increasingly relevant biological and clinical questions. Scientists are challenged in performing efficient and reproducible data extraction and analysis pipelines over heterogeneously processed datasets. Available software packages are suitable for analyzing experimental files from such datasets one by one, but do not scale to thousands of experiments. Moreover, they lack proper support for metadata manipulation. RESULTS: We present PyGMQL, a novel software for the manipulation of region-based genomic files and their relative metadata, built on top of the GMQL genomic big data management system. PyGMQL provides a set of expressive functions for the manipulation of region data and their metadata that can scale to arbitrary clusters and implicitly apply to thousands of files, producing millions of regions. PyGMQL provides data interoperability, distribution transparency and query outsourcing. The PyGMQL package integrates scalable data extraction over the Apache Spark engine underlying the GMQL implementation with native Python support for interactive data analysis and visualization. It supports data interoperability, solving the impedance mismatch between executing set-oriented queries and programming in Python. PyGMQL provides distribution transparency (the ability to address a remote dataset) and query outsourcing (the ability to assign processing to a remote service) in an orthogonal way. Outsourced processing can address cloud-based installations of the GMQL engine. CONCLUSIONS: PyGMQL is an effective and innovative tool for supporting tertiary data extraction and analysis pipelines. We demonstrate the expressiveness and performance of PyGMQL through a sequence of biological data analysis scenarios of increasing complexity, which highlight reproducibility, expressive power and scalability.


Assuntos
Análise de Dados , Bases de Dados Genéticas , Genômica , Software , Elementos Facilitadores Genéticos/genética , Genoma , Estudo de Associação Genômica Ampla , Humanos , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
9.
Nat Commun ; 10(1): 4749, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628347

RESUMO

Trophectoderm (TE) lineage development is pivotal for proper implantation, placentation, and healthy pregnancy. However, only a few TE-specific transcription factors (TFs) have been systematically characterized, hindering our understanding of the process. To elucidate regulatory mechanisms underlying TE development, here we map super-enhancers (SEs) in trophoblast stem cells (TSCs) as a model. We find both prominent TE-specific master TFs (Cdx2, Gata3, and Tead4), and >150 TFs that had not been previously implicated in TE lineage, that are SE-associated. Mapping targets of 27 SE-predicted TFs reveals a highly intertwined transcriptional regulatory circuitry. Intriguingly, SE-predicted TFs show 4 distinct expression patterns with dynamic alterations of their targets during TSC differentiation. Furthermore, depletion of a subset of TFs results in dysregulation of the markers for specialized cell types in placenta, suggesting a role during TE differentiation. Collectively, we characterize an expanded TE-specific regulatory network, providing a framework for understanding TE lineage development and placentation.


Assuntos
Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Trofoblastos/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Camundongos , Placentação/genética , Gravidez , Fatores de Transcrição/genética , Trofoblastos/citologia
10.
Endocrinology ; 160(12): 2877-2891, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31599948

RESUMO

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the control of phosphate (P) homeostasis and vitamin D metabolism. Despite advances, however, molecular details of this gene's regulation remain uncertain. In this report, we created mouse strains in which four epigenetically marked FGF23 regulatory regions were individually deleted from the mouse genome using CRISPR/Cas9 gene-editing technology, and the consequences of these mutations were then assessed on Fgf23 expression and regulation in vivo. An initial analysis confirmed that bone expression of Fgf23 and circulating intact FGF23 (iFGF23) were strongly influenced by both chronic dietary P treatment and acute injection of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, further analysis revealed that bone Fgf23 expression and iFGF23 could be rapidly upregulated by dietary P within 3 and 6 hours, respectively; this acute upregulation was lost in the FGF23-PKO mouse containing an Fgf23 proximal enhancer deletion but not in the additional enhancer-deleted mice. Of note, prolonged dietary P treatment over several days led to normalization of FGF23 levels in the FGF23-PKO mouse, suggesting added complexity associated with P regulation of FGF23. Treatment with 1,25(OH)2D3 also revealed a similar loss of Fgf23 induction and blood iFGF23 levels in this mouse. Finally, normal lipopolysaccharide (LPS) induction of Fgf23 expression was also compromised in the FGF23-PKO mouse, a result that, together with our previous report, indicates that the action of LPS on Fgf23 expression is mediated by both proximal and distal Fgf23 enhancers. These in vivo data provide key functional insight into the genomic enhancers through which Fgf23 expression is mediated.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Animais , Osso e Ossos/metabolismo , Sistemas CRISPR-Cas , Calcitriol , Elementos Facilitadores Genéticos , Feminino , Fatores de Crescimento de Fibroblastos/genética , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatos/sangue , Regiões Promotoras Genéticas
11.
BMC Bioinformatics ; 20(1): 474, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521109

RESUMO

BACKGROUND: In most mammals, a vast array of genes coding for chemosensory receptors mediates olfaction. Odorant receptor (OR) genes generally constitute the largest multifamily (> 1100 intact members in the mouse). From the whole pool, each olfactory neuron expresses a single OR allele following poorly characterized mechanisms termed OR gene choice. OR genes are found in genomic aggregations known as clusters. Nearby enhancers, named elements, are crucial regulators of OR gene choice. Despite their importance, searching for new elements is burdensome. Other chemosensory receptor genes responsible for smell adhere to expression modalities resembling OR gene choice, and are arranged in genomic clusters - often with chromosomal linkage to OR genes. Still, no elements are known for them. RESULTS: Here we present an inexpensive framework aimed at predicting elements. We redefine cluster identity by focusing on multiple receptor gene families at once, and exemplify thirty - not necessarily OR-exclusive - novel candidate enhancers. CONCLUSIONS: The pipeline we introduce could guide future in vivo work aimed at discovering/validating new elements. In addition, our study provides an updated and comprehensive classification of all genomic loci responsible for the transduction of olfactory signals in mammals.


Assuntos
Algoritmos , Elementos Facilitadores Genéticos , Genômica/métodos , Receptores Odorantes/genética , Análise de Sequência de DNA/normas , Animais , Humanos , Camundongos , Ratos
12.
Nucleic Acids Res ; 47(19): 10115-10133, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31555818

RESUMO

Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.


Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Autorrenovação Celular/genética , Reprogramação Celular/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Mutação/genética , Proteína Homeobox Nanog/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Transfecção
13.
Genome Biol ; 20(1): 197, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31514731

RESUMO

BACKGROUND: Robustness and evolutionary stability of gene expression in the human genome are established by an array of redundant enhancers. RESULTS: Using Hi-C data in multiple cell lines, we report a comprehensive map of promoters and active enhancers connected by chromatin contacts, spanning 9000 enhancer chains in 4 human cell lines associated with 2600 human genes. We find that the first enhancer in a chain that directly contacts the target promoter is commonly located at a greater genomic distance from the promoter than the second enhancer in a chain, 96 kb vs. 45 kb, respectively. The first enhancer also features higher similarity to the promoter in terms of tissue specificity and higher enrichment of loop factors, suggestive of a stable primary contact with the promoter. In contrast, a chain of enhancers which connects to the target promoter through a neutral DNA segment instead of an enhancer is associated with a significant decrease in target gene expression, suggesting an important role of the first enhancer in initiating transcription using the target promoter and bridging the promoter with other regulatory elements in the locus. CONCLUSIONS: The widespread chained structure of gene enhancers in humans reveals that the primary, critical enhancer is distal, commonly located further away than other enhancers. This first, distal enhancer establishes contacts with multiple regulatory elements and safeguards a complex regulatory program of its target gene.


Assuntos
Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Linhagem Celular , Cromatina/química , Regulação da Expressão Gênica , Humanos
14.
Nat Commun ; 10(1): 4154, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515496

RESUMO

To better understand the impact of chromatin structure on regulation of the prostate cancer transcriptome, we develop high-resolution chromatin interaction maps in normal and prostate cancer cells using in situ Hi-C. By combining the in situ Hi-C data with active and repressive histone marks, CTCF binding sites, nucleosome-depleted regions, and transcriptome profiling, we identify topologically associating domains (TADs) that change in size and epigenetic states between normal and prostate cancer cells. Moreover, we identify normal and prostate cancer-specific enhancer-promoter loops and involved transcription factors. For example, we show that FOXA1 is enriched in prostate cancer-specific enhancer-promoter loop anchors. We also find that the chromatin structure surrounding the androgen receptor (AR) locus is altered in the prostate cancer cells with many cancer-specific enhancer-promoter loops. This creation of 3D epigenomic maps enables a better understanding of prostate cancer biology and mechanisms of gene regulation.


Assuntos
Epigenômica , Neoplasias da Próstata/genética , Transcriptoma/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Loci Gênicos , Código das Histonas/genética , Humanos , Masculino , Regiões Promotoras Genéticas , Receptores Androgênicos/genética
16.
Nat Cell Biol ; 21(10): 1179-1190, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548608

RESUMO

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.


Assuntos
Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/metabolismo
17.
Nat Commun ; 10(1): 4221, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31530818

RESUMO

HiChIP/PLAC-seq is increasingly becoming popular for profiling 3D chromatin contacts among regulatory elements and for annotating functions of genetic variants. Here we describe FitHiChIP, a computational method for loop calling from HiChIP/PLAC-seq data, which jointly models the non-uniform coverage and genomic distance scaling of contact counts to compute statistical significance estimates. We also develop a technique to filter putative bystander loops that can be explained by stronger adjacent loops. Compared to existing methods, FitHiChIP performs better in recovering contacts reported by Hi-C, promoter capture Hi-C and ChIA-PET experiments and in capturing previously validated promoter-enhancer interactions. FitHiChIP loop calls are reproducible among replicates and are consistent across different experimental settings. Our work also provides a framework for differential HiChIP analysis with an option to utilize ChIP-seq data for further characterizing differential loops. Even though designed for HiChIP, FitHiChIP is also applicable to other conformation capture assays.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Cromatina/metabolismo , Biologia Computacional/métodos , Animais , Sítios de Ligação , Cromatina/genética , Elementos Facilitadores Genéticos , Genômica , Camundongos , Regiões Promotoras Genéticas
18.
BMC Med Genomics ; 12(1): 128, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500627

RESUMO

BACKGROUND: Genome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) that may be genetic factors underlying Alzheimer's disease (AD). However, how these AD-associated SNPs (AD SNPs) contribute to the pathogenesis of this disease is poorly understood because most of them are located in non-coding regions, such as introns and intergenic regions. Previous studies reported that some disease-associated SNPs affect regulatory elements including enhancers. We hypothesized that non-coding AD SNPs are located in enhancers and affect gene expression levels via chromatin loops. METHODS: To characterize AD SNPs within non-coding regions, we extracted 406 AD SNPs with GWAS p-values of less than 1.00 × 10- 6 from the GWAS catalog database. Of these, we selected 392 SNPs within non-coding regions. Next, we checked whether those non-coding AD SNPs were located in enhancers that typically regulate gene expression levels using publicly available data for enhancers that were predicted in 127 human tissues or cell types. We sought expression quantitative trait locus (eQTL) genes affected by non-coding AD SNPs within enhancers because enhancers are regulatory elements that influence the gene expression levels. To elucidate how the non-coding AD SNPs within enhancers affect the gene expression levels, we identified chromatin-chromatin interactions by Hi-C experiments. RESULTS: We report the following findings: (1) nearly 30% of non-coding AD SNPs are located in enhancers; (2) eQTL genes affected by non-coding AD SNPs within enhancers are associated with amyloid beta clearance, synaptic transmission, and immune responses; (3) 95% of the AD SNPs located in enhancers co-localize with their eQTL genes in topologically associating domains suggesting that regulation may occur through chromatin higher-order structures; (4) rs1476679 spatially contacts the promoters of eQTL genes via CTCF-CTCF interactions; (5) the effect of other AD SNPs such as rs7364180 is likely to be, at least in part, indirect through regulation of transcription factors that in turn regulate AD associated genes. CONCLUSION: Our results suggest that non-coding AD SNPs may affect the function of enhancers thereby influencing the expression levels of surrounding or distant genes via chromatin loops. This result may explain how some non-coding AD SNPs contribute to AD pathogenesis.


Assuntos
Doença de Alzheimer/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Variação Genética , Conformação de Ácido Nucleico , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Cromatina/química , Elementos Facilitadores Genéticos , Humanos , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética
19.
Nat Neurosci ; 22(10): 1718-1730, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501571

RESUMO

Activity-driven transcription plays an important role in many brain processes, including those underlying memory and epilepsy. Here we combine genetic tagging of nuclei and ribosomes with RNA sequencing, chromatin immunoprecipitation with sequencing, assay for transposase-accessible chromatin using sequencing and Hi-C to investigate transcriptional and chromatin changes occurring in mouse hippocampal excitatory neurons at different time points after synchronous activation during seizure and sparse activation by novel context exploration. The transcriptional burst is associated with an increase in chromatin accessibility of activity-regulated genes and enhancers, de novo binding of activity-regulated transcription factors, augmented promoter-enhancer interactions and the formation of gene loops that bring together the transcription start site and transcription termination site of induced genes and may sustain the fast reloading of RNA polymerase complexes. Some chromatin occupancy changes and interactions, particularly those driven by AP1, remain long after neuronal activation and could underlie the changes in neuronal responsiveness and circuit connectivity observed in these neuroplasticity paradigms, perhaps thereby contributing to metaplasticity in the adult brain.


Assuntos
Epigenômica , Hipocampo/fisiologia , Neurônios/fisiologia , Animais , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Genes Precoces/genética , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Regiões Promotoras Genéticas/genética , Convulsões/genética , Convulsões/fisiopatologia , Estado Epiléptico/genética , Estado Epiléptico/fisiopatologia , Fator de Transcrição AP-1/genética , Transcrição Genética/genética , Transcrição Genética/fisiologia
20.
Nat Genet ; 51(9): 1369-1379, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477927

RESUMO

Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell type-specific transcriptomes is not fully understood. We developed a simple and robust method, native elongating transcript-cap analysis of gene expression (NET-CAGE), to sensitively detect 5' ends of nascent RNAs in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer-promoter pairs are generally activated simultaneously on stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 atlas of transcribed enhancers, with broad applicability to biomedical research.


Assuntos
Regiões 5' não Traduzidas/genética , Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA/genética , Transcrição Genética , Perfilação da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Sítio de Iniciação de Transcrição , Transcriptoma
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