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1.
Am J Hum Genet ; 108(2): 257-268, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545029

RESUMO

Genome-wide chromatin conformation capture technologies such as Hi-C are commonly employed to study chromatin spatial organization. In particular, to identify statistically significant long-range chromatin interactions from Hi-C data, most existing methods such as Fit-Hi-C/FitHiC2 and HiCCUPS assume that all chromatin interactions are statistically independent. Such an independence assumption is reasonable at low resolution (e.g., 40 kb bin) but is invalid at high resolution (e.g., 5 or 10 kb bins) because spatial dependency of neighboring chromatin interactions is non-negligible at high resolution. Our previous hidden Markov random field-based methods accommodate spatial dependency but are computationally intensive. It is urgent to develop approaches that can model spatial dependence in a computationally efficient and scalable manner. Here, we develop HiC-ACT, an aggregated Cauchy test (ACT)-based approach, to improve the detection of chromatin interactions by post-processing results from methods assuming independence. To benchmark the performance of HiC-ACT, we re-analyzed deeply sequenced Hi-C data from a human lymphoblastoid cell line, GM12878, and mouse embryonic stem cells (mESCs). Our results demonstrate advantages of HiC-ACT in improving sensitivity with controlled type I error. By leveraging information from neighboring chromatin interactions, HiC-ACT enhances the power to detect interactions with lower signal-to-noise ratio and similar (if not stronger) epigenetic signatures that suggest regulatory roles. We further demonstrate that HiC-ACT peaks show higher overlap with known enhancers than Fit-Hi-C/FitHiC2 peaks in both GM12878 and mESCs. HiC-ACT, effectively a summary statistics-based approach, is computationally efficient (∼6 min and ∼2 GB memory to process 25,000 pairwise interactions).


Assuntos
Cromatina/genética , Cromatina/metabolismo , Genômica/métodos , Animais , Linhagem Celular , Cromatina/química , Simulação por Computador , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos , Humanos , Camundongos , Conformação Molecular , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA
2.
Viruses ; 13(2)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530363

RESUMO

The transcription of the HIV-1 provirus results in only one type of transcript-full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.


Assuntos
HIV-1/genética , Processamento de RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Processamento Alternativo , Éxons , Regulação Viral da Expressão Gênica , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/química , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico , Latência Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo
3.
Nat Commun ; 12(1): 531, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483495

RESUMO

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two individual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation.


Assuntos
Núcleo Celular/genética , Cromatina/genética , Células Eritroides/metabolismo , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Células Cultivadas , Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Regulação da Expressão Gênica , Genômica/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA
4.
Nat Commun ; 12(1): 567, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495464

RESUMO

The regulatory elements controlling gene expression during acute inflammation are not fully elucidated. Here we report the identification of a set of NF-κB-bound elements and common chromatin landscapes underlying the acute inflammatory response across cell-types and mammalian species. Using primary vascular endothelial cells (human/mouse/bovine) treated with the pro-inflammatory cytokine, Tumor Necrosis Factor-α, we identify extensive (~30%) conserved orthologous binding of NF-κB to accessible, as well as nucleosome-occluded chromatin. Regions with the highest NF-κB occupancy pre-stimulation show dramatic increases in NF-κB binding and chromatin accessibility post-stimulation. These 'pre-bound' regions are typically conserved (~56%), contain multiple NF-κB motifs, are utilized by diverse cell types, and overlap rare non-coding mutations and common genetic variation associated with both inflammatory and cardiovascular phenotypes. Genetic ablation of conserved, 'pre-bound' NF-κB regions within the super-enhancer associated with the chemokine-encoding CCL2 gene and elsewhere supports the functional relevance of these elements.


Assuntos
Cromatina/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/genética , Inflamação/genética , NF-kappa B/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Doença Aguda , Animais , Sítios de Ligação/genética , Bovinos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Cromatina/metabolismo , Sequência Conservada/genética , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lógica , Camundongos , Modelos Genéticos , Ligação Proteica , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia
5.
Nat Commun ; 12(1): 135, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420081

RESUMO

Since most variants that impact polygenic disease phenotypes localize to non-coding genomic regions, understanding the consequences of regulatory element variants will advance understanding of human disease mechanisms. Here, we report that the systemic lupus erythematosus (SLE) risk variant rs2431697 as likely causal for SLE through disruption of a regulatory element, modulating miR-146a expression. Using epigenomic analysis, genome-editing and 3D chromatin structure analysis, we show that rs2431697 tags a cell-type dependent distal enhancer specific for miR-146a that physically interacts with the miR-146a promoter. NF-kB binds the disease protective allele in a sequence-specific manner, increasing expression of this immunoregulatory microRNA. Finally, CRISPR activation-based modulation of this enhancer in the PBMCs of SLE patients attenuates type I interferon pathway activation by increasing miR-146a expression. Our work provides a strategy to define non-coding RNA functional regulatory elements using disease-associated variants and provides mechanistic links between autoimmune disease risk genetic variation and disease etiology.


Assuntos
Epigênese Genética/imunologia , Lúpus Eritematoso Sistêmico/genética , MicroRNAs/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sistemas CRISPR-Cas , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Predisposição Genética para Doença , Técnicas de Genotipagem , Células HEK293 , Voluntários Saudáveis , Humanos , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/transplante , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Regiões Promotoras Genéticas , RNA-Seq , Transdução de Sinais/genética , Transdução de Sinais/imunologia
6.
PLoS One ; 15(12): e0228233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347517

RESUMO

RNA interference (RNAi) plays key roles in post-transcriptional and chromatin modification levels as well as regulates various eukaryotic gene expressions which are involved in stress responses, development and maintenance of genome integrity during developmental stages. The whole mechanism of RNAi pathway is directly involved with the gene-silencing process by the interaction of Dicer-Like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) gene families and their regulatory elements. However, these RNAi gene families and their sub-cellular locations, functional pathways and regulatory components were not extensively investigated in the case of economically and nutritionally important fruit plant sweet orange (Citrus sinensis L.). Therefore, in silico characterization, gene diversity and regulatory factor analysis of RNA silencing genes in C. sinensis were conducted by using the integrated bioinformatics approaches. Genome-wide comparison analysis based on phylogenetic tree approach detected 4 CsDCL, 8 CsAGO and 4 CsRDR as RNAi candidate genes in C. sinensis corresponding to the RNAi genes of model plant Arabidopsis thaliana. The domain and motif composition and gene structure analyses for all three gene families exhibited almost homogeneity within the same group members. The Gene Ontology enrichment analysis clearly indicated that the predicted genes have direct involvement into the gene-silencing and other important pathways. The key regulatory transcription factors (TFs) MYB, Dof, ERF, NAC, MIKC_MADS, WRKY and bZIP were identified by their interaction network analysis with the predicted genes. The cis-acting regulatory elements associated with the predicted genes were detected as responsive to light, stress and hormone functions. Furthermore, the expressed sequence tag (EST) analysis showed that these RNAi candidate genes were highly expressed in fruit and leaves indicating their organ specific functions. Our genome-wide comparison and integrated bioinformatics analyses provided some necessary information about sweet orange RNA silencing components that would pave a ground for further investigation of functional mechanism of the predicted genes and their regulatory factors.


Assuntos
Citrus sinensis/genética , Regulação da Expressão Gênica de Plantas/genética , Interferência de RNA/fisiologia , Proteínas Argonauta/genética , Simulação por Computador , Etiquetas de Sequências Expressas , Frutas/metabolismo , Perfilação da Expressão Gênica/métodos , Genes de Plantas/genética , Genoma de Planta/genética , Família Multigênica/genética , Filogenia , Proteínas de Plantas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribonuclease III/genética , Fatores de Transcrição/metabolismo
7.
Development ; 147(23)2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323375

RESUMO

The central nervous system hosts parenchymal macrophages, known as microglia, and non-parenchymal macrophages, collectively termed border-associated macrophages (BAMs). Microglia, but not BAMs, were reported to be absent in mice lacking a conserved Csf1r enhancer: the fms-intronic regulatory element (FIRE). However, it is unknown whether FIRE deficiency also impacts BAM arrival and/or maintenance. Here, we show that macrophages in the ventricular system of the brain, including Kolmer's epiplexus macrophages, are absent in Csf1r ΔFIRE/ΔFIRE mice. Stromal choroid plexus BAMs are also considerably reduced. During normal development, we demonstrate that intracerebroventricular macrophages arrive from embryonic day 10.5, and can traverse ventricular walls in embryonic slice cultures. In Csf1r ΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations.


Assuntos
Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos/genética , Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Sistema Nervoso Central/crescimento & desenvolvimento , Embrião de Mamíferos , Íntrons/genética , Camundongos , Microglia/metabolismo , Tecido Parenquimatoso/crescimento & desenvolvimento , Tecido Parenquimatoso/metabolismo , Sequências Reguladoras de Ácido Nucleico
8.
BMC Bioinformatics ; 21(1): 507, 2020 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-33160328

RESUMO

BACKGROUND: Enhancer-promoter interactions (EPIs) play key roles in transcriptional regulation and disease progression. Although several computational methods have been developed to predict such interactions, their performances are not satisfactory when training and testing data from different cell lines. Currently, it is still unclear what extent a across cell line prediction can be made based on sequence-level information. RESULTS: In this work, we present a novel Sequence-based method (called SEPT) to predict the enhancer-promoter interactions in new cell line by using the cross-cell information and Transfer learning. SEPT first learns the features of enhancer and promoter from DNA sequences with convolutional neural network (CNN), then designing the gradient reversal layer of transfer learning to reduce the cell line specific features meanwhile retaining the features associated with EPIs. When the locations of enhancers and promoters are provided in new cell line, SEPT can successfully recognize EPIs in this new cell line based on labeled data of other cell lines. The experiment results show that SEPT can effectively learn the latent import EPIs-related features between cell lines and achieves the best prediction performance in terms of AUC (the area under the receiver operating curves). CONCLUSIONS: SEPT is an effective method for predicting the EPIs in new cell line. Domain adversarial architecture of transfer learning used in SEPT can learn the latent EPIs shared features among cell lines from all other existing labeled data. It can be expected that SEPT will be of interest to researchers concerned with biological interaction prediction.


Assuntos
Redes Neurais de Computação , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Área Sob a Curva , Linhagem Celular , Humanos , Curva ROC
9.
Nat Commun ; 11(1): 5560, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144558

RESUMO

Cancers result from a set of genetic and epigenetic alterations. Most known oncogenes were identified by gain-of-function mutations in cancer, yet little is known about their epigenetic features. Through integrative analysis of 11,596 epigenomic profiles and mutations from >8200 tumor-normal pairs, we discover broad genic repression domains (BGRD) on chromatin as an epigenetic signature for oncogenes. A BGRD is a widespread enrichment domain of the repressive histone modification H3K27me3 and is further enriched with multiple other repressive marks including H3K9me3, H3K9me2, and H3K27me2. Further, BGRD displays widespread enrichment of repressed cis-regulatory elements. Shortening of BGRDs is linked to derepression of transcription. BGRDs at oncogenes tend to be conserved across normal cell types. Putative tumor-promoting genes and lncRNAs defined using BGRDs are experimentally verified as required for cancer phenotypes. Therefore, BGRDs play key roles in epigenetic regulation of cancer and provide a direction for mutation-independent discovery of oncogenes.


Assuntos
Cromatina/genética , Inativação Gênica , Oncogenes , Neoplasias da Mama/genética , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sequência Conservada/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Motivos de Nucleotídeos/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Elongação da Transcrição Genética , Iniciação da Transcrição Genética
10.
BMC Bioinformatics ; 21(1): 464, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33076821

RESUMO

BACKGROUND: Genome browsers are widely used for locating interesting genomic regions, but their interactive use is obviously limited to inspecting short genomic portions. An ideal interaction is to provide patterns of regions on the browser, and then extract other genomic regions over the whole genome where such patterns occur, ranked by similarity. RESULTS: We developed SimSearch, an optimized pattern-search method and an open source plugin for the Integrated Genome Browser (IGB), to find genomic region sets that are similar to a given region pattern. It provides efficient visual genome-wide analytics computation in large datasets; the plugin supports intuitive user interactions for selecting an interesting pattern on IGB tracks and visualizing the computed occurrences of similar patterns along the entire genome. SimSearch also includes functions for the annotation and enrichment of results, and is enhanced with a Quickload repository including numerous epigenomic feature datasets from ENCODE and Roadmap Epigenomics. The paper also includes some use cases to show multiple genome-wide analyses of biological interest, which can be easily performed by taking advantage of the presented approach. CONCLUSIONS: The novel SimSearch method provides innovative support for effective genome-wide pattern search and visualization; its relevance and practical usefulness is demonstrated through a number of significant use cases of biological interest. The SimSearch IGB plugin, documentation, and code are freely available at https://deib-geco.github.io/simsearch-app/ and https://github.com/DEIB-GECO/simsearch-app/ .


Assuntos
Algoritmos , Epigenômica , Genoma , Reconhecimento Automatizado de Padrão , Navegador , Humanos , Anotação de Sequência Molecular , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo
11.
Nucleic Acids Res ; 48(19): 10909-10923, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045748

RESUMO

The three-dimensional configuration of the chromatin architecture is known to be crucial for alterations in the transcriptional network; however, the underlying mechanisms of epigenetic control of senescence-related gene expression by modulating the chromatin architecture remain unknown. Here, we demonstrate frequent chromosomal compartment switching during mouse embryonic fibroblasts (MEFs) replicative senescence as characterized by senescence-inactivated (SIAEs) and -activated enhancers (SAEs) in topologically associated domains (TADs). Mechanistically, SAEs are closely correlated with senescence-associated secretory phenotype (SASP) genes, which are a key transcriptional feature of an aging microenvironment that contributes to tumor progression, aging acceleration, and immunoinflammatory responses. Moreover, SAEs can positively regulate robust changes in SASP expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is capable of enhancing SAE activity, which accelerates the emergence of SAEs flanking SASPs and the secretion of downstream factors, contributing to the progression of senescence. Our results provide novel insight into the TAD-related control of SASP gene expression by revealing hierarchical roles of the chromatin architecture, transcription factors, and enhancer activity in the regulation of cellular senescence.


Assuntos
Envelhecimento/genética , Senescência Celular , Fibroblastos/citologia , Regulação da Expressão Gênica , Animais , Células Cultivadas , Cromatina/metabolismo , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Sequências Reguladoras de Ácido Nucleico
12.
Proc Natl Acad Sci U S A ; 117(38): 23991-24000, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32879011

RESUMO

The genomic sequences of crops continue to be produced at a frenetic pace. It remains challenging to develop complete annotations of functional genes and regulatory elements in these genomes. Chromatin accessibility assays enable discovery of functional elements; however, to uncover the full portfolio of cis-elements would require profiling of many combinations of cell types, tissues, developmental stages, and environments. Here, we explore the potential to use DNA methylation profiles to develop more complete annotations. Using leaf tissue in maize, we define ∼100,000 unmethylated regions (UMRs) that account for 5.8% of the genome; 33,375 UMRs are found greater than 2 kb from genes. UMRs are highly stable in multiple vegetative tissues, and they capture the vast majority of accessible chromatin regions from leaf tissue. However, many UMRs are not accessible in leaf, and these represent regions with potential to become accessible in specific cell types or developmental stages. These UMRs often occur near genes that are expressed in other tissues and are enriched for binding sites of transcription factors. The leaf-inaccessible UMRs exhibit unique chromatin modification patterns and are enriched for chromatin interactions with nearby genes. The total UMR space in four additional monocots ranges from 80 to 120 megabases, which is remarkably similar considering the range in genome size of 271 megabases to 4.8 gigabases. In summary, based on the profile from a single tissue, DNA methylation signatures provide powerful filters to distill large genomes down to the small fraction of putative functional genes and regulatory elements.


Assuntos
Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Sequências Reguladoras de Ácido Nucleico/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA de Plantas/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Zea mays/genética
13.
BMC Bioinformatics ; 21(1): 416, 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32962625

RESUMO

BACKGROUND: Comparative genomics studies are growing in number partly because of their unique ability to provide insight into shared and divergent biology between species. Of particular interest is the use of phylogenetic methods to infer the evolutionary history of cis-regulatory sequence features, which contribute strongly to phenotypic divergence and are frequently gained and lost in eutherian genomes. Understanding the mechanisms by which cis-regulatory element turnover generate emergent phenotypes is crucial to our understanding of adaptive evolution. Ancestral reconstruction methods can place species-specific cis-regulatory features in their evolutionary context, thus increasing our understanding of the process of regulatory sequence turnover. However, applying these methods to gain and loss of cis-regulatory features historically required complex workflows, preventing widespread adoption by the broad scientific community. RESULTS: MapGL simplifies phylogenetic inference of the evolutionary history of short genomic sequence features by combining the necessary steps into a single piece of software with a simple set of inputs and outputs. We show that MapGL can reliably disambiguate the mechanisms underlying differential regulatory sequence content across a broad range of phylogenetic topologies and evolutionary distances. Thus, MapGL provides the necessary context to evaluate how genomic sequence gain and loss contribute to species-specific divergence. CONCLUSIONS: MapGL makes phylogenetic inference of species-specific sequence gain and loss easy for both expert and non-expert users, making it a powerful tool for gaining novel insights into genome evolution.


Assuntos
Evolução Molecular , Genoma/genética , Genômica/métodos , Sequências Reguladoras de Ácido Nucleico , Software , Animais , Humanos , Mamíferos/genética , Fenótipo , Filogenia
14.
Nucleic Acids Res ; 48(18): 10602-10613, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976557

RESUMO

Currently, predictive translation tuning of regulatory elements to the desired output of transcription factor (TF)-based biosensors remains a challenge. The gene expression of a biosensor system must exhibit appropriate translation intensity, which is controlled by the ribosome-binding site (RBS), to achieve fine-tuning of its dynamic range (i.e. fold change in gene expression between the presence and absence of inducer) by adjusting the translation level of the TF and reporter. However, existing TF-based biosensors generally suffer from unpredictable dynamic range. Here, we elucidated the connections and partial mechanisms between RBS, translation level, protein folding and dynamic range, and presented a design platform that predictably tuned the dynamic range of biosensors based on deep learning of large datasets cross-RBSs (cRBSs). In doing so, a library containing 7053 designed cRBSs was divided into five sub-libraries through fluorescence-activated cell sorting to establish a classification model based on convolutional neural network in deep learning. Finally, the present work exhibited a powerful platform to enable predictable translation tuning of RBS to the dynamic range of biosensors.


Assuntos
Técnicas Biossensoriais , Sequências Reguladoras de Ácido Nucleico/genética , Ribossomos/genética , Fatores de Transcrição/genética , Sítios de Ligação/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/isolamento & purificação
15.
Proc Natl Acad Sci U S A ; 117(41): 25655-25666, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32978299

RESUMO

Although we know many sequence-specific transcription factors (TFs), how the DNA sequence of cis-regulatory elements is decoded and orchestrated on the genome scale to determine immune cell differentiation is beyond our grasp. Leveraging a granular atlas of chromatin accessibility across 81 immune cell types, we asked if a convolutional neural network (CNN) could learn to infer cell type-specific chromatin accessibility solely from regulatory DNA sequences. With a tailored architecture and an ensemble approach to CNN parameter interpretation, we show that our trained network ("AI-TAC") does so by rediscovering ab initio the binding motifs for known regulators and some unknown ones. Motifs whose importance is learned virtually as functionally important overlap strikingly well with positions determined by chromatin immunoprecipitation for several TFs. AI-TAC establishes a hierarchy of TFs and their interactions that drives lineage specification and also identifies stage-specific interactions, like Pax5/Ebf1 vs. Pax5/Prdm1, or the role of different NF-κB dimers in different cell types. AI-TAC assigns Spi1/Cebp and Pax5/Ebf1 as the drivers necessary for myeloid and B lineage fates, respectively, but no factors seemed as dominantly required for T cell differentiation, which may represent a fall-back pathway. Mouse-trained AI-TAC can parse human DNA, revealing a strikingly similar ranking of influential TFs and providing additional support that AI-TAC is a generalizable regulatory sequence decoder. Thus, deep learning can reveal the regulatory syntax predictive of the full differentiative complexity of the immune system.


Assuntos
Aprendizado Profundo , Hematopoese/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição , Animais , Cromatina/genética , Bases de Dados Genéticas , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Fatores de Transcrição/química , Fatores de Transcrição/genética
16.
PLoS Genet ; 16(9): e1009023, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925947

RESUMO

Lung cancer is the leading cause of cancer-related death and lung adenocarcinoma is its most common subtype. Although genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations driving lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We found over 32,000 enhancers that appear differentially activated between normal lung and lung adenocarcinoma. Among the identified transcriptional regulators inactivated in lung adenocarcinoma vs. normal lung, NKX2-1 was linked to a large number of silenced enhancers. Among the activated transcriptional regulators identified, CENPA, FOXM1, and MYBL2 were linked to numerous cancer-specific enhancers. High expression of CENPA, FOXM1, and MYBL2 is particularly observed in a subgroup of lung adenocarcinomas and is associated with poor patient survival. Notably, CENPA, FOXM1, and MYBL2 are also key regulators of cancer-specific enhancers in breast adenocarcinoma of the basal subtype, but they are associated with distinct sets of activated enhancers. We identified individual lung adenocarcinoma enhancers linked to CENPA, FOXM1, or MYBL2 that were associated with poor patient survival. Knockdown experiments of FOXM1 and MYBL2 suggest that these factors regulate genes involved in controlling cell cycle progression and cell division. For example, we found that expression of TK1, a potential target gene of a MYBL2-linked enhancer, is associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma epigenomes, highlighting novel potential targets for clinical intervention.


Assuntos
Adenocarcinoma de Pulmão/genética , Epigênese Genética/genética , Elementos Reguladores de Transcrição/genética , Adenocarcinoma/genética , Adulto , Idoso , Proteínas de Ciclo Celular/genética , Epigenômica , Proteína Forkhead Box M1/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Homeobox , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Sequências Reguladoras de Ácido Nucleico/genética
17.
Yi Chuan ; 42(8): 760-774, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32952112

RESUMO

Gene expression and three-dimensional (3D) genome organization are thought to be closely related. The protocadherin (Pcdh) clusters are central for neuronal self-avoidance in brain development and have been used as model genes to explore the role of 3D genome structures in gene regulation. Transcription factor RFX5 (regulatory factor x 5) is a member of the winged-helix subfamily of the helix-turn-helix superfamily proteins. The RFX5 protein contains four domains: oligomerization, DNA-binding, helical, and transactivation domains. RFX5 plays an important role in regulating expression of the major histocompatibility complex class II (MHC II) gene complex. Here we report a role of RFX5 in the regulation of clustered Pcdh genes. We first knocked out the RFX5 gene by using CRISPR/Cas9 DNA-fragment editing in HEC-1-B cell line. By RNA-seq experiments, we found that RFX5 deletion results in a significant increase of expression levels of the Pcdhα6, Pcdhα12 and Pcdhαc2 genes. By ChIP-nexus experiments, we found that RFX5 deletion leads to the increased binding of CTCF and RAD21 in the Pcdhα cluster. Finally, through quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that RFX5 deletion results in a significant increase of long-distance chromatin interactions between the HS5-1 enhancer and its target promoters in the Pcdhα cluster. In conclusion, RFX5 regulates gene expression of the Pcdhα cluster through higher-order chromatin structure.


Assuntos
Caderinas , Regulação da Expressão Gênica , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição , Caderinas/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Humanos , Regiões Promotoras Genéticas/genética , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Sequências Reguladoras de Ácido Nucleico
18.
PLoS Genet ; 16(9): e1009019, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32915782

RESUMO

Loci identified in genome-wide association studies (GWAS) can include multiple distinct association signals. We sought to identify the molecular basis of multiple association signals for adiponectin, a hormone involved in glucose regulation secreted almost exclusively from adipose tissue, identified in the Metabolic Syndrome in Men (METSIM) study. With GWAS data for 9,262 men, four loci were significantly associated with adiponectin: ADIPOQ, CDH13, IRS1, and PBRM1. We performed stepwise conditional analyses to identify distinct association signals, a subset of which are also nearly independent (lead variant pairwise r2<0.01). Two loci exhibited allelic heterogeneity, ADIPOQ and CDH13. Of seven association signals at the ADIPOQ locus, two signals colocalized with adipose tissue expression quantitative trait loci (eQTLs) for three transcripts: trait-increasing alleles at one signal were associated with increased ADIPOQ and LINC02043, while trait-increasing alleles at the other signal were associated with decreased ADIPOQ-AS1. In reporter assays, adiponectin-increasing alleles at two signals showed corresponding directions of effect on transcriptional activity. Putative mechanisms for the seven ADIPOQ signals include a missense variant (ADIPOQ G90S), a splice variant, a promoter variant, and four enhancer variants. Of two association signals at the CDH13 locus, the first signal consisted of promoter variants, including the lead adipose tissue eQTL variant for CDH13, while a second signal included a distal intron 1 enhancer variant that showed ~2-fold allelic differences in transcriptional reporter activity. Fine-mapping and experimental validation demonstrated that multiple, distinct association signals at these loci can influence multiple transcripts through multiple molecular mechanisms.


Assuntos
Adiponectina/genética , Adiponectina/metabolismo , Tecido Adiposo/metabolismo , Alelos , Caderinas/genética , Caderinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Frequência do Gene/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Síndrome Metabólica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
PLoS Genet ; 16(8): e1008976, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866141

RESUMO

Neural circuitry for mating and reproduction resides within the terminal segments of central nervous system (CNS) which express Hox paralogous group 9-13 (in vertebrates) or Abdominal-B (Abd-B) in Drosophila. Terminal neuroblasts (NBs) in A8-A10 segments of Drosophila larval CNS are subdivided into two groups based on expression of transcription factor Doublesex (Dsx). While the sex specific fate of Dsx-positive NBs is well investigated, the fate of Dsx-negative NBs is not known so far. Our studies with Dsx-negative NBs suggests that these cells, like their abdominal counterparts (in A3-A7 segments) use Hox, Grainyhead (Grh) and Notch to undergo cell death during larval development. This cell death also happens by transcriptionally activating RHG family of apoptotic genes through a common apoptotic enhancer in early to mid L3 stages. However, unlike abdominal NBs (in A3-A7 segments) which use increasing levels of resident Hox factor Abdominal-A (Abd-A) as an apoptosis trigger, Dsx-negative NBs (in A8-A10 segments) keep the levels of resident Hox factor Abd-B constant. These cells instead utilize increasing levels of the temporal transcription factor Grh and a rise in Notch activity to gain apoptotic competence. Biochemical and in vivo analysis suggest that Abdominal-A and Grh binding motifs in the common apoptotic enhancer also function as Abdominal-B and Grh binding motifs and maintains the enhancer activity in A8-A10 NBs. Finally, the deletion of this enhancer by the CRISPR-Cas9 method blocks the apoptosis of Dsx-negative NBs. These results highlight the fact that Hox dependent NB apoptosis in abdominal and terminal regions utilizes common molecular players (Hox, Grh and Notch), but seems to have evolved different molecular strategies to pattern CNS.


Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Receptores Notch/genética , Fatores de Transcrição/genética , Abdome/crescimento & desenvolvimento , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Células-Tronco Neurais/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética
20.
Proc Natl Acad Sci U S A ; 117(32): 19544-19555, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32747566

RESUMO

Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. Here, we identify a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) in Drosophila This network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function are directed by cis-regulatory elements of developmental control genes that include homologs of mammalian Zinc finger of the cerebellum and Purkinje cell protein 4 Drosophila DTB-specific cis-regulatory elements correspond to regulatory sequences of human ENGRAILED-2, PAX-2, and DACHSHUND-1 that direct MHB-specific expression in the embryonic mouse brain. We show that cis-regulatory elements and the gene networks they regulate direct the formation and function of midbrain circuits for balance and motor coordination in insects and mammals. Regulatory mechanisms mediating the genetic specification of cephalic neural circuits in arthropods correspond to those in chordates, thereby implying their origin before the divergence of deuterostomes and ecdysozoans.


Assuntos
Evolução Molecular , Redes Reguladoras de Genes , Mesencéfalo/fisiologia , Animais , Comportamento Animal , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Drosophila , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Sequências Reguladoras de Ácido Nucleico , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Rombencéfalo/fisiologia , Transdução de Sinais
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