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1.
Mol Biol (Mosk) ; 53(3): 393-401, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184604

RESUMO

The efficiency at which specific transcription factors interact with DNA may vary in the presence of single nucleotide polymorphisms (SNPs), and the variation provides an important mechanism that regulates expression of human genes and contributes to the individual susceptibility to various diseases. Ample genetic and epigenetic data make it possible to predict both functional polymorphic variants and the transcription factors whose binding they affect. However, predictions of the kind require experimental verification. An original method developed for the purpose includes immunoprecipitation of DNA-protein complexes, followed by quantification of the bound DNA by real-time PCR. The method does not require chemical modification of the DNA probes and yield reproducible results with total nuclear extracts from cultured human cells.


Assuntos
Alelos , Imunoprecipitação , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Humanos , Ligação Proteica
2.
Nat Commun ; 10(1): 2633, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201330

RESUMO

Long-range chromatin interactions are important for transcriptional regulation of genes, many of which are related to complex agronomics traits. However, the pattern of three-dimensional chromatin interactions remains unclear in plants. Here we report the generation of chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data and the construction of extensive H3K4me3- and H3K27ac-centered chromatin interaction maps in maize. Results show that the interacting patterns between proximal and distal regulatory regions of genes are highly complex and dynamic. Genes with chromatin interactions have higher expression levels than those without interactions. Genes with proximal-proximal interactions prefer to be transcriptionally coordinated. Tissue-specific proximal-distal interactions are associated with tissue-specific expression of genes. Interactions between proximal and distal regulatory regions further interweave into organized network communities that are enriched in specific biological functions. The high-resolution chromatin interaction maps will help to understand the transcription regulation of genes associated with complex agronomic traits of maize.


Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Genética/genética , Zea mays/genética , Cromatina/genética , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Produção Agrícola , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Histonas/genética , Histonas/imunologia , Regiões Promotoras Genéticas , Locos de Características Quantitativas/genética
3.
Nat Methods ; 16(5): 397-400, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962623

RESUMO

We present cisTopic, a probabilistic framework used to simultaneously discover coaccessible enhancers and stable cell states from sparse single-cell epigenomics data ( http://github.com/aertslab/cistopic ). Using a compendium of single-cell ATAC-seq datasets from differentiating hematopoietic cells, brain and transcription factor perturbations, we demonstrate that topic modeling can be exploited for robust identification of cell types, enhancers and relevant transcription factors. cisTopic provides insight into the mechanisms underlying regulatory heterogeneity in cell populations.


Assuntos
Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Modelos Teóricos , Análise de Célula Única/métodos , Animais , Células Sanguíneas/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Análise por Conglomerados , Redes Reguladoras de Genes/genética , Humanos , Camundongos , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de RNA , Fluxo de Trabalho
4.
Methods Cell Biol ; 151: 219-235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948010

RESUMO

Programs of gene transcription are controlled by cis-acting DNA elements, including enhancers, silencers, and promoters. Local accessibility of chromatin has proven to be a highly informative structural feature for identifying such regulatory elements, which tend to be relatively open due to their interactions with proteins. Recently, ATAC-seq (assay for transposase-accessible chromatin using sequencing) has emerged as one of the most powerful approaches for genome-wide chromatin accessibility profiling. This method assesses DNA accessibility using hyperactive Tn5 transposase, which simultaneously cuts DNA and inserts sequencing adaptors, preferentially in regions of open chromatin. ATAC-seq is a relatively simple procedure which can be applied to only a few thousand cells. It is well-suited to developing embryos of sea urchins and other echinoderms, which are a prominent experimental model for understanding the genomic control of animal development. In this chapter, we present a protocol for applying ATAC-seq to embryonic cells of sea urchins.


Assuntos
Cromatina/genética , Equinodermos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Animais , Equinodermos/crescimento & desenvolvimento , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Elementos Silenciadores Transcricionais/genética , Transposases/química , Transposases/genética
5.
Gene Expr Patterns ; 32: 53-66, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30940554

RESUMO

We have cloned and characterized an intronic fragment of zebrafish lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) that drives expression to the zebrafish enveloping layer (EVL). L-plastin is a calcium-dependent actin-bundling protein belonging to the plastin/fimbrin family of proteins, and is necessary for the proper migration and attachment of several adult cell types, including leukocytes and osteoclasts. However, in zebrafish lcp1 is abundantly expressed much earlier, during differentiation of the EVL. The cells of this epithelial layer migrate collectively, spreading vegetally over the yolk. L-plastin expression persists into the larval periderm, a transient epithelial tissue that forms the first larval skin. This finding establishes that L-plastin is activated in two different embryonic waves, with a distinct regulatory switch between the early EVL and the later leukocyte. To better study L-plastin expressing cells we attempted CRISPR/Cas9 homology-driven recombination (HDR) to insert a self-cleaving peptide (Cre-P2A-EGFP-CAAX) downstream of the native lcp1 promoter. This produced a stable zebrafish line expressing Cre recombinase in EVL nuclei and green fluorescence in EVL cell membranes. In vivo tracking of these labeled cells provided enhanced views of EVL migration behavior, membrane extensions, and mitotic events. Finally, we experimentally dissected key elements of the targeted lcp1 locus, discovering a ∼300 bp intronic sequence sufficient to drive EVL expression. The lcp1: Cre-P2A-EGFP-CAAX zebrafish should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the conserved regulatory sequences we have isolated predict that L-plastin orthologs may have a similar early expression pattern in other vertebrate embryos.


Assuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Embrião não Mamífero/metabolismo , Epitélio/crescimento & desenvolvimento , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Sequências Reguladoras de Ácido Nucleico/genética , Transcriptoma/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
6.
Int J Mol Sci ; 20(7)2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959806

RESUMO

Abstract: Deciphering the code of cis-regulatory element (CRE) is one of the core issues of current biology. As an important category of CRE, enhancers play crucial roles in gene transcriptional regulations in a distant manner. Further, the disruption of an enhancer can cause abnormal transcription and, thus, trigger human diseases, which means that its accurate identification is currently of broad interest. Here, we introduce an innovative concept, i.e., abelian complexity function (ACF), which is a more complex extension of the classic subword complexity function, for a new coding of DNA sequences. After feature selection by an upper bound estimation and integration with DNA composition features, we developed an enhancer prediction model with hybrid abelian complexity features (HACF). Compared with existing methods, HACF shows consistently superior performance on three sources of enhancer datasets. We tested the generalization ability of HACF by scanning human chromosome 22 to validate previously reported super-enhancers. Meanwhile, we identified novel candidate enhancers which have supports from enhancer-related ENCODE ChIP-seq signals. In summary, HACF improves current enhancer prediction and may be beneficial for further prioritization of functional noncoding variants.


Assuntos
Biologia Computacional/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Algoritmos , Sequência de Bases , Cromossomos Humanos Par 22/genética , Doença/genética , Elementos Facilitadores Genéticos , Entropia , Éxons/genética , Humanos , Íntrons/genética , Regiões Promotoras Genéticas/genética
7.
Nat Genet ; 51(4): 611-617, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30926969

RESUMO

Transposable elements (TEs) are an abundant and rich genetic resource of regulatory sequences1-3. Cryptic regulatory elements within TEs can be epigenetically reactivated in cancer to influence oncogenesis in a process termed onco-exaptation4. However, the prevalence and impact of TE onco-exaptation events across cancer types are poorly characterized. Here, we analyzed 7,769 tumors and 625 normal datasets from 15 cancer types, identifying 129 TE cryptic promoter-activation events involving 106 oncogenes across 3,864 tumors. Furthermore, we interrogated the AluJb-LIN28B candidate: the genetic deletion of the TE eliminated oncogene expression, while dynamic DNA methylation modulated promoter activity, illustrating the necessity and sufficiency of a TE for oncogene activation. Collectively, our results characterize the global profile of TE onco-exaptation and highlight this prevalent phenomenon as an important mechanism for promiscuous oncogene activation and ultimately tumorigenesis.


Assuntos
Elementos de DNA Transponíveis/genética , Neoplasias/genética , Oncogenes/genética , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA/genética , Evolução Molecular , Células HEK293 , Humanos , Células K562 , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética
8.
Mol Cell ; 74(2): 245-253.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30826165

RESUMO

Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , DNA/química , Conformação de Ácido Nucleico , Proteínas Repressoras/química , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , DNA/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Motivos de Nucleotídeos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Repressoras/genética
9.
Nat Commun ; 10(1): 1209, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872577

RESUMO

Sparse profiling of CpG methylation in blood by microarrays has identified epigenetic links to common diseases. Here we apply methylC-capture sequencing (MCC-Seq) in a clinical population of ~200 adipose tissue and matched blood samples (Ntotal~400), providing high-resolution methylation profiling (>1.3 M CpGs) at regulatory elements. We link methylation to cardiometabolic risk through associations to circulating plasma lipid levels and identify lipid-associated CpGs with unique localization patterns in regulatory elements. We show distinct features of tissue-specific versus tissue-independent lipid-linked regulatory regions by contrasting with parallel assessments in ~800 independent adipose tissue and blood samples from the general population. We follow-up on adipose-specific regulatory regions under (1) genetic and (2) epigenetic (environmental) regulation via integrational studies. Overall, the comprehensive sequencing of regulatory element methylomes reveals a rich landscape of functional variants linked genetically as well as epigenetically to plasma lipid traits.


Assuntos
Doenças Cardiovasculares/genética , Ilhas de CpG/genética , Epigênese Genética , Doenças Metabólicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Tecido Adiposo/metabolismo , Adulto , Idoso , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/metabolismo , Metilação de DNA , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lipídeos/sangue , Masculino , Doenças Metabólicas/sangue , Doenças Metabólicas/metabolismo , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos
10.
Gene ; 699: 54-61, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30858133

RESUMO

Epigenetic regulatory changes alter the gene regulation function of DNA repeat elements in cancer and consequently promote malignant phenotypes. Some short tandem repeat sequences, distributed throughout the human genome, can play a role as cis-regulatory elements of the genes. Distributions of tandem long (≥10) and short (<10) A-T repeats in the genome are different depending on gene functions. Long repeats are more commonly found in housekeeping genes and may regulate genes in harmonious fashion. Mononucleotide A-repeats around transcription start sites interact with Argonaute proteins (AGOs) to regulate gene expression. miRNA-bound AGO alterations in cancer have been reported; consequently, these changes would affect genes containing mononucleotide A- and T-repeats. Here, we showed an unprecedented hallmark of gene regulation in cancer. We evaluated the gene expression profiles reported in the Gene Expression Omnibus and found a high density of 13-27 A-T repeats in the up-regulated genes in malignancies derived from the bladder, cervix, head and neck, ovary, vulva, breast, colon, liver, lung, prostate, kidney, thyroid, adrenal gland, bone, blood cells, muscle and brain. Transfection of cell-penetrating protein tag AGO1 containing poly uracils (CPP-AGO1-polyUs) to the lung cancer cell lines altered gene regulation depending on the presence of long A-T repeats. CPP-AGO1-polyUs limited cell proliferation and the ability of a cancer cell to grow into a colony in lung cancer cell lines. In conclusion, long A-T repeats up-regulated many genes in cancer that can be targeted by AGO1 to change the expression of many genes and limited cancer growth.


Assuntos
Proteínas Argonauta/genética , Fatores de Iniciação em Eucariotos/genética , Repetições de Microssatélites/genética , Neoplasias/genética , Transcrição Genética/genética , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , MicroRNAs/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sítio de Iniciação de Transcrição/fisiologia , Transcriptoma/genética , Regulação para Cima/genética
11.
Nat Genet ; 51(4): 683-693, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858613

RESUMO

Widespread linkage disequilibrium and incomplete annotation of cell-to-cell state variation represent substantial challenges to elucidating mechanisms of trait-associated genetic variation. Here we perform genetic fine-mapping for blood cell traits in the UK Biobank to identify putative causal variants. These variants are enriched in genes encoding proteins in trait-relevant biological pathways and in accessible chromatin of hematopoietic progenitors. For regulatory variants, we explore patterns of developmental enhancer activity, predict molecular mechanisms, and identify likely target genes. In several instances, we localize multiple independent variants to the same regulatory element or gene. We further observe that variants with pleiotropic effects preferentially act in common progenitor populations to direct the production of distinct lineages. Finally, we leverage fine-mapped variants in conjunction with continuous epigenomic annotations to identify trait-cell type enrichments within closely related populations and in single cells. Our study provides a comprehensive framework for single-variant and single-cell analyses of genetic associations.


Assuntos
Hematopoese/genética , Polimorfismo de Nucleotídeo Único/genética , Linhagem da Célula/genética , Cromatina/genética , Mapeamento Cromossômico/métodos , Epigenômica/métodos , Estudo de Associação Genômica Ampla/métodos , Humanos , Desequilíbrio de Ligação/genética , Fenótipo , Locos de Características Quantitativas/genética , Sequências Reguladoras de Ácido Nucleico/genética
12.
Genetics ; 211(4): 1239-1254, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30796012

RESUMO

Conserved noncoding elements (CNEs) have a significant regulatory influence on their neighboring genes. Loss of proximity to CNEs through genomic rearrangements can, therefore, impact the transcriptional states of the cognate genes. Yet, the evolutionary implications of such chromosomal alterations have not been studied. Through genome-wide analysis of CNEs and the cognate genes of representative species from five different mammalian orders, we observed a significant loss of genes' linear proximity to CNEs in the rat lineage. The CNEs and the genes losing proximity had a significant association with fetal, but not postnatal, brain development as assessed through ontology terms, developmental gene expression, chromatin marks, and genetic mutations. The loss of proximity to CNEs correlated with the independent evolutionary loss of fetus-specific upregulation of nearby genes in the rat brain. DNA breakpoints implicated in brain abnormalities of germline origin had significant representation between a CNE and the gene that exhibited loss of proximity, signifying the underlying developmental tolerance of genomic rearrangements that allowed the evolutionary splits of CNEs and the cognate genes in the rodent lineage. Our observations highlighted a nontrivial impact of chromosomal rearrangements in shaping the evolutionary dynamics of mammalian brain development and might explain the loss of brain traits, like cerebral folding of the cortex, in the rodent lineage.


Assuntos
Encéfalo/metabolismo , Sequência Conservada , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Encéfalo/embriologia , Bovinos , Cães , Rearranjo Gênico , Cavalos , Humanos , Neurogênese , Ratos
13.
Proc Natl Acad Sci U S A ; 116(9): 3668-3677, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755522

RESUMO

Histones are modified by enzymes that act in a locus, cell-type, and developmental stage-specific manner. The recruitment of enzymes to chromatin is regulated at multiple levels, including interaction with sequence-specific DNA-binding factors. However, the DNA-binding specificity of the regulatory factors that orchestrate specific histone modifications has not been broadly mapped. We have analyzed 6 histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3, K3H9me3, H3K36me3) across 121 human cell types and tissues from the NIH Roadmap Epigenomics Project as well as 8 histone marks (with addition of H3K4me2 and H3K9ac) from the mouse ENCODE Consortium. We have identified 361 and 369 DNA motifs in human and mouse, respectively, that are the most predictive of each histone mark. Interestingly, 107 human motifs are conserved between the two species. In human embryonic cell line H1, we mutated only the found DNA motifs at particular loci and the significant reduction of H3K27ac levels validated the regulatory roles of the perturbed motifs. The functionality of these motifs was also supported by the evidence that histone-associated motifs, especially H3K4me3 motifs, significantly overlap with the expression of quantitative trait loci SNPs in cancer patients more than the known and random motifs. Furthermore, we observed possible feedbacks to control chromatin dynamics as the found motifs appear in the promoters or enhancers associated with various histone modification enzymes. These results pave the way toward revealing the molecular mechanisms of epigenetic events, such as histone modification dynamics and epigenetic priming.


Assuntos
Metilação de DNA/genética , Código das Histonas/genética , Motivos de Nucleotídeos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Cromatina/genética , Proteínas de Ligação a DNA/genética , Epigenômica , Humanos , Camundongos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional/genética
14.
Nucleic Acids Res ; 47(5): 2306-2321, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30605520

RESUMO

RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including super-enhancers and repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.


Assuntos
DNA/química , DNA/isolamento & purificação , Conformação de Ácido Nucleico , RNA/química , RNA/isolamento & purificação , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , DNA/genética , Elementos Facilitadores Genéticos/genética , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Purinas/química , Purinas/metabolismo , RNA/genética , RNA Longo não Codificante/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
15.
Nat Commun ; 10(1): 335, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30659195

RESUMO

Hepatocellular carcinomas (HCC) exhibit distinct promoter hypermethylation patterns, but the epigenetic regulation and function of transcriptional enhancers remain unclear. Here, our affinity- and bisulfite-based whole-genome sequencing analyses reveal global enhancer hypomethylation in human HCCs. Integrative epigenomic characterization further pinpoints a recurrent hypomethylated enhancer of CCAAT/enhancer-binding protein-beta (C/EBPß) which correlates with C/EBPß over-expression and poorer prognosis of patients. Demethylation of C/EBPß enhancer reactivates a self-reinforcing enhancer-target loop via direct transcriptional up-regulation of enhancer RNA. Conversely, deletion of this enhancer via CRISPR/Cas9 reduces C/EBPß expression and its genome-wide co-occupancy with BRD4 at H3K27ac-marked enhancers and super-enhancers, leading to drastic suppression of driver oncogenes and HCC tumorigenicity. Hepatitis B X protein transgenic mouse model of HCC recapitulates this paradigm, as C/ebpß enhancer hypomethylation associates with oncogenic activation in early tumorigenesis. These results support a causal link between aberrant enhancer hypomethylation and C/EBPß over-expression, thereby contributing to hepatocarcinogenesis through global transcriptional reprogramming.


Assuntos
Carcinogênese/genética , Metilação de DNA , Neoplasias Hepáticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desmetilação , Epigênese Genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Fígado , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Transativadores , Fatores de Transcrição/metabolismo , Ativação Transcricional , Regulação para Cima
16.
Nat Commun ; 10(1): 470, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30692544

RESUMO

Integrative analysis of multi-omics layers at single cell level is critical for accurate dissection of cell-to-cell variation within certain cell populations. Here we report scCAT-seq, a technique for simultaneously assaying chromatin accessibility and the transcriptome within the same single cell. We show that the combined single cell signatures enable accurate construction of regulatory relationships between cis-regulatory elements and the target genes at single-cell resolution, providing a new dimension of features that helps direct discovery of regulatory patterns specific to distinct cell identities. Moreover, we generate the first single cell integrated map of chromatin accessibility and transcriptome in early embryos and demonstrate the robustness of scCAT-seq in the precise dissection of master transcription factors in cells of distinct states. The ability to obtain these two layers of omics data will help provide more accurate definitions of "single cell state" and enable the deconvolution of regulatory heterogeneity from complex cell populations.


Assuntos
Cromatina/genética , Epigenômica , Regulação da Expressão Gênica , Análise de Célula Única/métodos , Transcriptoma , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células HCT116 , Células HeLa , Humanos , Células K562 , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA/métodos
17.
Gut ; 68(5): 928-941, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30692146

RESUMO

Genome-wide association studies have identified over 200 loci associated with IBD. We and others have recently shown that, in addition to variants in protein-coding genes, the majority of the associated loci are related to DNA regulatory elements (DREs). These findings add a dimension to the already complex genetic background of IBD. In this review we summarise the existing evidence on the role of DREs in IBD. We discuss how epigenetic research can be used in candidate gene approaches that take non-coding variants into account and can help to pinpoint the essential pathways and cell types in the pathogenesis of IBD. Despite the increased level of genetic complexity, these findings can contribute to novel therapeutic options that target transcription factor binding and enhancer activity. Finally, we summarise the future directions and challenges of this emerging field.


Assuntos
Epigenômica , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/terapia , Sequências Reguladoras de Ácido Nucleico/genética , Humanos
18.
Nat Genet ; 51(2): 343-353, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30692680

RESUMO

Loci discovered by genome-wide association studies predominantly map outside protein-coding genes. The interpretation of the functional consequences of non-coding variants can be greatly enhanced by catalogs of regulatory genomic regions in cell lines and primary tissues. However, robust and readily applicable methods are still lacking by which to systematically evaluate the contribution of these regions to genetic variation implicated in diseases or quantitative traits. Here we propose a novel approach that leverages genome-wide association studies' findings with regulatory or functional annotations to classify features relevant to a phenotype of interest. Within our framework, we account for major sources of confounding not offered by current methods. We further assess enrichment of genome-wide association studies for 19 traits within Encyclopedia of DNA Elements- and Roadmap-derived regulatory regions. We characterize unique enrichment patterns for traits and annotations driving novel biological insights. The method is implemented in standalone software and an R package, to facilitate its application by the research community.


Assuntos
Doença/genética , Genoma/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Anotação de Sequência Molecular/métodos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Software
19.
Gene ; 688: 132-139, 2019 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-30529096

RESUMO

To investigate whether the members of the mammalian Achaete-Scute Complex homologue (ASH) gene family have evolved functional differences, we used the patterning of bristles as a phenotypic marker. Drosophila uses a single genetic locus - the Achaete-Scute Complex - to demarcate the regions of the body where bristles can form. We found 4-5 Achaete-Scute Complex homologue genes (ASH) in the mammalian genome, which are homologous with scute in Drosophila. Although ASH2 and ASH3 have gained new functions during evolution, the function of ASH4 and its evolutionary changes are still unclear. In this study, we overexpressed mouse and human ASH1 and ASH4 in the Drosophila notum respectively. The results show that both the protein sequence and cis-regulatory elements of mammalian ASH1 have conserved an ancient proneural function during evolution. However, mouse ASH4 has lost proneural function partly due to truncation of a C-terminal amino acid domain. Interestingly, instead of a similar loss of proneural function, we found human ASH4 can actually inhibit Drosophila bristle development, implying that human ASH4 may be a potential factor relating to skin development in human being. Our results demonstrate gene duplication of the ASH family may have led to a novel function during evolution.


Assuntos
Drosophila/genética , Mamíferos/genética , Neurogênese/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Evolução Biológica , Padronização Corporal/genética , Sequência Conservada , Duplicação Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos
20.
Dev Biol ; 446(2): 168-179, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30594505

RESUMO

Cis-regulatory elements are critical for the precise spatiotemporal regulation of genes during development. However, identifying functional regulatory sites that drive cell differentiation in vivo has been complicated by the high numbers of cells required for whole-genome epigenetic assays. Here, we identified putative regulatory elements during sex determination by performing ATAC-seq and ChIP-seq for H3K27ac in purified XX and XY gonadal supporting cells before and after sex determination in mice. We show that XX and XY supporting cells initiate sex determination with similar chromatin landscapes and acquire sex-specific regulatory elements as they commit to the male or female fate. To validate our approach, we identified a functional gonad-specific enhancer downstream of Bmp2, an ovary-promoting gene. This work increases our understanding of the complex regulatory network underlying mammalian sex determination and provides a powerful resource for identifying non-coding regulatory elements that could harbor mutations that lead to Disorders of Sexual Development.


Assuntos
Cromatina/genética , Gônadas/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Processos de Determinação Sexual/genética , Acetilação , Animais , Cromatina/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/citologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos
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