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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(11): 1236-1240, 2020 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-33179228

RESUMO

OBJECTIVE: To explore the effect of rare synonymous variants of the ATP7B gene on the splicing of its precursor mRNA. METHODS: A total of 248 rare synonymous variants with allelic frequency of <0.005 were retrieved from the ExAc database. Human Splicing Finder (HSF) was used to predict their effect on the splicing of precursor mRNA. And ESE Finder 3.0 was used to predict the effect of such variants on the binding ability of SR protein family. Rare synonymous variants affecting the binding of two or more SR proteins were selected and verified with an in vitro mini gene splicing report system. RESULTS: HSF analysis indicated that 136 of the 248 rare synonymous variants may destroy the exonic splicing enhancer (ESE) motif. Analysis using ESE Finder 3.0 indicated that 19 of them may affect the binding of two or more SR proteins at the same time. In vitro mini gene experiment confirmed that the c.1620C>T (p.L540L) and c.3888C>T (p.A1296A) variants could lead to abnormal splicing of the corresponding exons, resulting in complete skipping of exon 4 and 25% increase in the skipping of exon 18, respectively. CONCLUSION: Synonymous variants may affect the splicing of precursor mRNA in various ways, particularly the destruction of ESE motif. This study confirmed that the c.1620C>T (p.L540L) and c.3888C>T (p.A1296A) variants can affect the mRNA splicing of the ATP7B gene, resulting in skipping of corresponding exons, which may provide a basis for genetic diagnosis and consultation of carriers.


Assuntos
Processamento Alternativo , ATPases Transportadoras de Cobre/genética , Elementos Facilitadores Genéticos , Éxons , Frequência do Gene , Humanos , RNA Mensageiro/genética
2.
Nat Commun ; 11(1): 4912, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999275

RESUMO

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, key tissues and cell-types required for functional inference are absent from large-scale resources. Here we explore the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using data from 420 donors. We find: (a) 7741 cis-eQTLs in islets with a replication rate across 44 GTEx tissues between 40% and 73%; (b) marked overlap between islet cis-eQTL signals and active regulatory sequences in islets, with reduced eQTL effect size observed in the stretch enhancers most strongly implicated in GWAS signal location; (c) enrichment of islet cis-eQTL signals with T2D risk variants identified in genome-wide association studies; and (d) colocalization between 47 islet cis-eQTLs and variants influencing T2D or glycemic traits, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in disease relevant tissues.


Assuntos
Glicemia/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Ilhotas Pancreáticas/metabolismo , Locos de Características Quantitativas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Glicemia/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Diabetes Mellitus Tipo 2/sangue , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA-Seq , Análise de Sequência de DNA , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Adulto Jovem
3.
Nat Commun ; 11(1): 5061, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033262

RESUMO

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.


Assuntos
Reprogramação Celular , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Catálise , Proliferação de Células , Senescência Celular , Desmetilação , Elementos Facilitadores Genéticos/genética , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Ativação Transcricional/genética
4.
Mol Cell ; 80(2): 359-373.e8, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32991830

RESUMO

Eukaryotic gene expression regulation involves thousands of distal regulatory elements. Understanding the quantitative contribution of individual enhancers to gene expression is critical for assessing the role of disease-associated genetic risk variants. Yet, we lack the ability to accurately link genes with their distal regulatory elements. To address this, we used 3D enhancer-promoter (E-P) associations identified using split-pool recognition of interactions by tag extension (SPRITE) to build a predictive model of gene expression. Our model dramatically outperforms models using genomic proximity and can be used to determine the quantitative impact of enhancer loss on gene expression in different genetic backgrounds. We show that genes that form stable E-P hubs have less cell-to-cell variability in gene expression. Finally, we identified transcription factors that regulate stimulation-dependent E-P interactions. Together, our results provide a framework for understanding quantitative contributions of E-P interactions and associated genetic variants to gene expression.


Assuntos
Bactérias/isolamento & purificação , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Animais , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica , Modelos Lineares , Camundongos Endogâmicos C57BL , Modelos Biológicos , Processos Estocásticos , Fatores de Transcrição/metabolismo
5.
Cell Rep ; 32(13): 108199, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32966801

RESUMO

ACE2 binds the coronavirus SARS-CoV-2 and facilitates its cellular entry. Interferons activate ACE2 expression in pneumocytes, suggesting a critical role of cytokines in SARS-CoV-2 target cells. Viral RNA was detected in breast milk in at least seven studies, raising the possibility that ACE2 is expressed in mammary tissue during lactation. Here, we show that Ace2 expression in mouse mammary tissue is induced during pregnancy and lactation, which coincides with the activation of intronic enhancers. These enhancers are occupied by the prolactin-activated transcription factor STAT5 and additional regulatory factors, including RNA polymerase II. Deletion of Stat5a results in decommissioning of the enhancers and an 83% reduction of Ace2 mRNA. We also demonstrate that Ace2 expression increases during lactation in lung, but not in kidney and intestine. JAK/STAT components are present in a range of SARS-CoV-2 target cells, opening the possibility that cytokines contribute to the viral load and extrapulmonary pathophysiology.


Assuntos
Janus Quinases/metabolismo , Peptidil Dipeptidase A/metabolismo , Gravidez/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Elementos Facilitadores Genéticos , Feminino , Humanos , Mucosa Intestinal/metabolismo , Rim/metabolismo , Lactação/metabolismo , Pulmão/metabolismo , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/genética , Fator de Transcrição STAT5/genética , Transdução de Sinais
6.
Nat Commun ; 11(1): 4544, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917861

RESUMO

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Loci Gênicos/genética , Ativação Transcricional , Acetilação , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Eritroblastos , Feminino , Feto , Código das Histonas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , RNA-Seq
7.
Yi Chuan ; 42(9): 817-831, 2020 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-32952117

RESUMO

Enhancers are a type of cis-acting DNA elements that enhance transcriptional activity of target genes. However, the uncertainty in the orientation and distance between enhancers and target genes could post significant difficulties in identifying the target genes and the regulatory mechanisms of the enhancers. Numerous studies have shown that the mutations and/or abnormalities in the functions of enhancers are associated with development of diseases. A few studies have reported that enhancers could activate cancer development or drug resistance by promoting the expression of target genes. At present, enhancers involved in carcinogenesis and drug resistance have not been fully identified, and the underlying mechanism are still largely unknown. This paper summarizes the main methods used in identifying and characterizing enhancers and analyzing the regulatory mechanism at the genome-wide level. It further reviews the recent research progress of enhancers in cancer diagnosis, treatment, and the underlying mechanism during carcinogenesis, thereby providing a reference for the screening of these enhancers involved in carcinogenesis and drug resistance and exploring their regulatory mechanisms of target genes. It also provides a new perspective for improving the diagnosis of cancer and insights for formulating cancer therapeutic strategies.


Assuntos
Elementos Facilitadores Genéticos , Neoplasias , Carcinogênese , Humanos , Neoplasias/genética
8.
Nat Commun ; 11(1): 4402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879318

RESUMO

Genome-wide association studies have identified genetic variation contributing to complex disease risk. However, assigning causal genes and mechanisms has been more challenging because disease-associated variants are often found in distal regulatory regions with cell-type specific behaviours. Here, we collect ATAC-seq, Hi-C, Capture Hi-C and nuclear RNA-seq data in stimulated CD4+ T cells over 24 h, to identify functional enhancers regulating gene expression. We characterise changes in DNA interaction and activity dynamics that correlate with changes in gene expression, and find that the strongest correlations are observed within 200 kb of promoters. Using rheumatoid arthritis as an example of T cell mediated disease, we demonstrate interactions of expression quantitative trait loci with target genes, and confirm assigned genes or show complex interactions for 20% of disease associated loci, including FOXO1, which we confirm using CRISPR/Cas9.


Assuntos
Artrite Reumatoide/genética , Linfócitos T CD4-Positivos/metabolismo , Cromatina , Proteína Forkhead Box O1/genética , Doenças Autoimunes/genética , Linfócitos T CD4-Positivos/citologia , Cromatina/química , Cromatina/genética , Elementos Facilitadores Genéticos , Proteína Forkhead Box O1/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Cultura Primária de Células , Regiões Promotoras Genéticas , Locos de Características Quantitativas
9.
Nucleic Acids Res ; 48(17): 9550-9570, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32810208

RESUMO

Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Neurônios/fisiologia , RNA/fisiologia , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Humanos , Neurônios/citologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Imagem Individual de Molécula
10.
Nucleic Acids Res ; 48(17): 9621-9636, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32853367

RESUMO

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra-Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Cromatina/genética , Regulação da Expressão Gênica , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Proteínas de Homeodomínio/genética , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Timo/citologia
11.
Proc Natl Acad Sci U S A ; 117(35): 21450-21458, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817427

RESUMO

How overall principles of cell-type-specific gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type-specific regulation employs different cis elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer-promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type-specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.


Assuntos
Fatores Etários , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Cromatina , Elementos Facilitadores Genéticos/genética , Eritroblastos , Eritropoese/fisiologia , Feminino , Expressão Gênica , Genômica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética
12.
Science ; 369(6503): 561-565, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732423

RESUMO

Most neuropsychiatric disease risk variants are in noncoding sequences and lack functional interpretation. Because regulatory sequences often reside in open chromatin, we reasoned that neuropsychiatric disease risk variants may affect chromatin accessibility during neurodevelopment. Using human induced pluripotent stem cell (iPSC)-derived neurons that model developing brains, we identified thousands of genetic variants exhibiting allele-specific open chromatin (ASoC). These neuronal ASoCs were partially driven by altered transcription factor binding, overrepresented in brain gene enhancers and expression quantitative trait loci, and frequently associated with distal genes through chromatin contacts. ASoCs were enriched for genetic variants associated with brain disorders, enabling identification of functional schizophrenia risk variants and their cis-target genes. This study highlights ASoC as a functional mechanism of noncoding neuropsychiatric risk variants, providing a powerful framework for identifying disease causal variants and genes.


Assuntos
Alelos , Encéfalo/metabolismo , Cromatina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Esquizofrenia/genética , Elementos Facilitadores Genéticos , Humanos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Risco
13.
Nat Commun ; 11(1): 4136, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811816

RESUMO

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Fator de Transcrição GATA3/química , Nucleossomos/química , Nucleossomos/genética , Motivos de Aminoácidos/genética , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/ultraestrutura , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Dedos de Zinco/genética
14.
PLoS Genet ; 16(8): e1008752, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790717

RESUMO

Myelin is composed of plasma membrane spirally wrapped around axons and compacted into dense sheaths by myelin-associated proteins. Myelin is elaborated by neuroepithelial derived oligodendrocytes in the central nervous system (CNS) and by neural crest derived Schwann cells in the peripheral nervous system (PNS). While some myelin proteins accumulate in only one lineage, myelin basic protein (Mbp) is expressed in both. Overlapping the Mbp gene is Golli, a transcriptional unit that is expressed widely both within and beyond the nervous system. A super-enhancer domain within the Golli/Mbp locus contains multiple enhancers shown previously to drive reporter construct expression specifically in oligodendrocytes or Schwann cells. In order to determine the contribution of each enhancer to the Golli/Mbp expression program, and to reveal if functional interactions occur among them, we derived mouse lines in which they were deleted, either singly or in different combinations, and relative mRNA accumulation was measured at key stages of early development and at maturity. Although super-enhancers have been shown previously to facilitate interaction among their component enhancers, the enhancers investigated here demonstrated largely additive relationships. However, enhancers demonstrating autonomous activity strictly in one lineage, when missing, were found to significantly reduce output in the other, thus revealing cryptic "stealth" activity. Further, in the absence of a key oligodendrocyte enhancer, Golli accumulation was markedly and uniformly attenuated in all cell types investigated. Our observations suggest a model in which enhancer-mediated DNA-looping and potential super-enhancer properties underlie Golli/Mbp regulatory organization.


Assuntos
Elementos Facilitadores Genéticos , Proteína Básica da Mielina/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/metabolismo , Neurogênese , Oligodendroglia/citologia , Oligodendroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(34): 20636-20644, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32778581

RESUMO

The diversity of forms in multicellular organisms originates largely from the spatial redeployment of developmental genes [S. B. Carroll, Cell 134, 25-36 (2008)]. Several scenarios can explain the emergence of cis-regulatory elements that govern novel aspects of a gene expression pattern [M. Rebeiz, M. Tsiantis, Curr. Opin. Genet. Dev. 45, 115-123 (2017)]. One scenario, enhancer co-option, holds that a DNA sequence producing an ancestral regulatory activity also becomes the template for a new regulatory activity, sharing regulatory information. While enhancer co-option might fuel morphological diversification, it has rarely been documented [W. J. Glassford et al., Dev. Cell 34, 520-531 (2015)]. Moreover, if two regulatory activities are borne from the same sequence, their modularity, considered a defining feature of enhancers [J. Banerji, L. Olson, W. Schaffner, Cell 33, 729-740 (1983)], might be affected by pleiotropy. Sequence overlap may thereby play a determinant role in enhancer function and evolution. Here, we investigated this problem with two regulatory activities of the Drosophila gene yellow, the novel spot enhancer and the ancestral wing blade enhancer. We used precise and comprehensive quantification of each activity in Drosophila wings to systematically map their sequences along the locus. We show that the spot enhancer has co-opted the sequences of the wing blade enhancer. We also identified a pleiotropic site necessary for DNA accessibility of a shared regulatory region. While the evolutionary steps leading to the derived activity are still unknown, such pleiotropy suggests that enhancer accessibility could be one of the molecular mechanisms seeding evolutionary co-option.


Assuntos
Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Evolução Biológica , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Evolução Molecular , Elementos Reguladores de Transcrição/genética , Asas de Animais/metabolismo
16.
PLoS One ; 15(8): e0233673, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750050

RESUMO

Computational algorithms are often used to assess pathogenicity of Variants of Uncertain Significance (VUS) that are found in disease-associated genes. Most computational methods include analysis of protein multiple sequence alignments (PMSA), assessing interspecies variation. Careful validation of PMSA-based methods has been done for relatively few genes, partially because creation of curated PMSAs is labor-intensive. We assessed how PMSA-based computational tools predict the effects of the missense changes in the APC gene, in which pathogenic variants cause Familial Adenomatous Polyposis. Most Pathogenic or Likely Pathogenic APC variants are protein-truncating changes. However, public databases now contain thousands of variants reported as missense. We created a curated APC PMSA that contained >3 substitutions/site, which is large enough for statistically robust in silico analysis. The creation of the PMSA was not easily automated, requiring significant querying and computational analysis of protein and genome sequences. Of 1924 missense APC variants in the NCBI ClinVar database, 1800 (93.5%) are reported as VUS. All but two missense variants listed as P/LP occur at canonical splice or Exonic Splice Enhancer sites. Pathogenicity predictions by five computational tools (Align-GVGD, SIFT, PolyPhen2, MAPP, REVEL) differed widely in their predictions of Pathogenic/Likely Pathogenic (range 17.5-75.0%) and Benign/Likely Benign (range 25.0-82.5%) for APC missense variants in ClinVar. When applied to 21 missense variants reported in ClinVar and securely classified as Benign, the five methods ranged in accuracy from 76.2-100%. Computational PMSA-based methods can be an excellent classifier for variants of some hereditary cancer genes. However, there may be characteristics of the APC gene and protein that confound the results of in silico algorithms. A systematic study of these features could greatly improve the automation of alignment-based techniques and the use of predictive algorithms in hereditary cancer genes.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Genes APC , Mutação de Sentido Incorreto , Algoritmos , Sequência de Aminoácidos , Biologia Computacional/métodos , Simulação por Computador , Bases de Dados de Proteínas , Elementos Facilitadores Genéticos , Evolução Molecular , Éxons , Variação Genética , Humanos , Filogenia , Isoformas de Proteínas/genética , Sítios de Splice de RNA , Alinhamento de Sequência/estatística & dados numéricos
17.
Nat Commun ; 11(1): 3920, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764605

RESUMO

How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development.


Assuntos
Deficiências do Desenvolvimento/genética , Epigênese Genética , Organogênese/genética , Animais , Animais Geneticamente Modificados , Bases de Dados Genéticas , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Código das Histonas/genética , Humanos , Modelos Genéticos , Mutação , Organogênese/fisiologia , Regiões Promotoras Genéticas , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética
18.
Mol Cell Biol ; 40(21)2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32839292

RESUMO

Differentiation status of tumors is correlated with metastatic potential and malignancy. FOXA1 (forkhead box A1) is a transcription factor known to regulate differentiation in certain tissues. Here, we investigate FOXA1 function in human colorectal cancer (CRC). We found that FOXA1 is robustly expressed in the normal human colon but significantly downregulated in colon adenocarcinoma. Applying FOXA1 chromatin immunoprecipitation coupled with deep sequencing and transcriptome analysis upon FOXA1 knockdown in well-differentiated CRC cells and FOXA1 overexpression in poorly differentiated CRC cells, we identified novel protein-coding and lncRNA genes regulated by FOXA1. Among the numerous novel FOXA1 targets we identified, we focused on CEACAM5, a tumor marker and facilitator of cell adhesion. We show that FOXA1 binds to a distal enhancer downstream of CEACAM5 and strongly activates its expression. Consistent with these data, we show that FOXA1 inhibits anoikis in CRC cells. Collectively, our results uncover novel protein-coding and noncoding targets of FOXA1 and suggest a vital role of FOXA1 in enhancing CEACAM5 expression and anoikis resistance in CRC cells.


Assuntos
Neoplasias Colorretais/genética , Redes Reguladoras de Genes , Fator 3-alfa Nuclear de Hepatócito/genética , RNA Longo não Codificante/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Anoikis/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Neoplasias Colorretais/patologia , Elementos Facilitadores Genéticos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas/genética , Pseudogenes
19.
Nat Genet ; 52(8): 811-818, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32632335

RESUMO

We developed cis-X, a computational method for discovering regulatory noncoding variants in cancer by integrating whole-genome and transcriptome sequencing data from a single cancer sample. cis-X first finds aberrantly cis-activated genes that exhibit allele-specific expression accompanied by an elevated outlier expression. It then searches for causal noncoding variants that may introduce aberrant transcription factor binding motifs or enhancer hijacking by structural variations. Analysis of 13 T-lineage acute lymphoblastic leukemias identified a recurrent intronic variant predicted to cis-activate the TAL1 oncogene, a finding validated in vivo by chromatin immunoprecipitation sequencing of a patient-derived xenograft. Candidate oncogenes include the prolactin receptor PRLR activated by a focal deletion that removes a CTCF-insulated neighborhood boundary. cis-X may be applied to pediatric and adult solid tumors that are aneuploid and heterogeneous. In contrast to existing approaches, which require large sample cohorts, cis-X enables the discovery of regulatory noncoding variants in individual cancer genomes.


Assuntos
Elementos Facilitadores Genéticos/genética , Variação Genética/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA não Traduzido/genética , Adolescente , Alelos , Criança , Pré-Escolar , Cromatina/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Oncogenes/genética , Transcrição Genética/genética
20.
BMC Bioinformatics ; 21(1): 317, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32689977

RESUMO

BACKGROUND: The binding sites of transcription factors (TFs) and the localisation of histone modifications in the human genome can be quantified by the chromatin immunoprecipitation assay coupled with next-generation sequencing (ChIP-seq). The resulting chromatin feature data has been successfully adopted for genome-wide enhancer identification by several unsupervised and supervised machine learning methods. However, the current methods predict different numbers and different sets of enhancers for the same cell type and do not utilise the pattern of the ChIP-seq coverage profiles efficiently. RESULTS: In this work, we propose a PRobabilistic Enhancer PRedictIoN Tool (PREPRINT) that assumes characteristic coverage patterns of chromatin features at enhancers and employs a statistical model to account for their variability. PREPRINT defines probabilistic distance measures to quantify the similarity of the genomic query regions and the characteristic coverage patterns. The probabilistic scores of the enhancer and non-enhancer samples are utilised to train a kernel-based classifier. The performance of the method is demonstrated on ENCODE data for two cell lines. The predicted enhancers are computationally validated based on the transcriptional regulatory protein binding sites and compared to the predictions obtained by state-of-the-art methods. CONCLUSION: PREPRINT performs favorably to the state-of-the-art methods, especially when requiring the methods to predict a larger set of enhancers. PREPRINT generalises successfully to data from cell type not utilised for training, and often the PREPRINT performs better than the previous methods. The PREPRINT enhancers are less sensitive to the choice of prediction threshold. PREPRINT identifies biologically validated enhancers not predicted by the competing methods. The enhancers predicted by PREPRINT can aid the genome interpretation in functional genomics and clinical studies.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Genoma Humano , Genômica/métodos , Histonas/genética , Modelos Estatísticos , Fatores de Transcrição/metabolismo , Cromatina/química , Cromatina/metabolismo , Código das Histonas , Histonas/química , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional
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