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1.
PLoS Comput Biol ; 15(11): e1007337, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682597

RESUMO

Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing.


Assuntos
Diferenciação Celular/genética , Ensaios de Triagem em Larga Escala/métodos , Elementos Reguladores de Transcrição/genética , Animais , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Bases de Dados Genéticas , Epigênese Genética , Epigenômica , Regulação da Expressão Gênica/genética , Genoma , Macrófagos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo
2.
PLoS Comput Biol ; 15(11): e1007399, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682602

RESUMO

Genome segmentation approaches allow us to characterize regulatory states in a given cell type using combinatorial patterns of histone modifications and other regulatory signals. In order to analyze regulatory state differences across cell types, current genome segmentation approaches typically require that the same regulatory genomics assays have been performed in all analyzed cell types. This necessarily limits both the numbers of cell types that can be analyzed and the complexity of the resulting regulatory states, as only a small number of histone modifications have been profiled across many cell types. Data imputation approaches that aim to estimate missing regulatory signals have been applied before genome segmentation. However, this approach is computationally costly and propagates any errors in imputation to produce incorrect genome segmentation results downstream. We present an extension to the IDEAS genome segmentation platform which can perform genome segmentation on incomplete regulatory genomics dataset collections without using imputation. Instead of relying on imputed data, we use an expectation-maximization approach to estimate marginal density functions within each regulatory state. We demonstrate that our genome segmentation results compare favorably with approaches based on imputation or other strategies for handling missing data. We further show that our approach can accurately impute missing data after genome segmentation, reversing the typical order of imputation/genome segmentation pipelines. Finally, we present a new 2D genome segmentation analysis of 127 human cell types studied by the Roadmap Epigenomics Consortium. By using an expanded set of chromatin marks that have been profiled in subsets of these cell types, our new segmentation results capture a more complex picture of combinatorial regulatory patterns that appear on the human genome.


Assuntos
Previsões/métodos , Elementos Reguladores de Transcrição/genética , Análise de Sequência de DNA/métodos , Algoritmos , Epigenômica , Redes Reguladoras de Genes/genética , Genoma Humano , Genômica/métodos , Humanos , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico , Sequenciamento Completo do Genoma/métodos
3.
Mol Cell Biol ; 39(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31591143

RESUMO

DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate the cis-acting responses to DSBs in G1 phase cells. We found that DSBs within a gene body silence its expression, as well as the transcription of local undamaged genes at a distance defined by the spread of γ-H2AX from the DSB. Importantly, DSBs not only repress ongoing transcription but also block the inducible expression of regional genes. DSB-mediated transcriptional repression depends on DDR signaling but does not require the generation of inaccessible chromatin. Our findings demonstrate that in G1 phase cells, DDR signaling establishes a robust and extensive region of transcriptional repression spreading from DSB sites and introduce an approach to study the mechanistic impact of targeted DNA breaks in nearly any chromatin environment.


Assuntos
Reparo do DNA/genética , Fase G1/genética , Elementos Silenciadores Transcricionais/genética , Animais , Ciclo Celular/genética , Linhagem Celular , DNA/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/fisiologia , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fase G1/fisiologia , Humanos , Camundongos , Elementos Reguladores de Transcrição/genética , Elementos Reguladores de Transcrição/fisiologia , Elementos Silenciadores Transcricionais/fisiologia
4.
Nat Genet ; 51(10): 1506-1517, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570892

RESUMO

The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell-derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes.


Assuntos
Fibrilação Atrial/genética , Fibrilação Atrial/patologia , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Elementos Reguladores de Transcrição , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Criança , Eletrocardiografia , Epigenômica , Feminino , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Ligação Proteica , Transcriptoma , Adulto Jovem
5.
Nat Commun ; 10(1): 4698, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619674

RESUMO

T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4+ T lymphocytes, with CRISPR/Cas9-guided deletion of these genomic segments supporting their role in RORγt expression. Mechanistically, T cell receptor stimulation induces cyclosporine A-sensitive histone modifications and P300/CBP acetylase recruitment at these elements in activated CD4+ T cells. Meanwhile, NFAT proteins bind to these regulatory elements and activate RORγt transcription in cooperation with NF-kB. Our data thus demonstrate that NFAT specifically regulate RORγt expression by binding to the RORC locus and promoting its permissive conformation.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição NFATC/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Elementos Reguladores de Transcrição/genética , Células Th17/metabolismo , Timócitos/metabolismo , Ativação Transcricional , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem da Célula , Citometria de Fluxo , Células HEK293 , Código das Histonas , Humanos , Células Jurkat , Células Th17/citologia , Timócitos/citologia , Fatores de Transcrição de p300-CBP/metabolismo
6.
PLoS Genet ; 15(10): e1008444, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589607

RESUMO

Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Homeostase/genética , Fatores de Transcrição/metabolismo , Asas de Animais/embriologia , Animais , Animais Geneticamente Modificados , Sítios de Ligação/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Genes de Insetos/fisiologia , Proteínas de Homeodomínio/genética , Larva/crescimento & desenvolvimento , Mutação , Elementos Reguladores de Transcrição/fisiologia , Fatores de Transcrição/genética
7.
EMBO J ; 38(20): e102161, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31531882

RESUMO

Differentiation of normal and tumor cells is controlled by regulatory networks enforced by lineage-determining transcription factors (TFs). Among them, TFs such as FOXA1/2 bind naïve chromatin and induce its accessibility, thus establishing new gene regulatory networks. Pancreatic ductal adenocarcinoma (PDAC) is characterized by the coexistence of well- and poorly differentiated cells at all stages of disease. How the transcriptional networks determining such massive cellular heterogeneity are established remains to be determined. We found that FOXA2, a TF controlling pancreas specification, broadly contributed to the cis-regulatory networks of PDACs. Despite being expressed in both well- and poorly differentiated PDAC cells, FOXA2 displayed extensively different genomic distributions and controlled distinct gene expression programs. Grade-specific functions of FOXA2 depended on its partnership with TFs whose expression varied depending on the differentiation grade. These data suggest that FOXA2 contributes to the regulatory networks of heterogeneous PDAC cells via interactions with alternative partner TFs.


Assuntos
Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Fator 1-beta Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias Pancreáticas/patologia , Elementos Reguladores de Transcrição , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Movimento Celular , Proliferação de Células , Redes Reguladoras de Genes , Fator 1-beta Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Humanos , Gradação de Tumores , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Células Tumorais Cultivadas
8.
Int J Mol Sci ; 20(18)2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31540007

RESUMO

Members of the plant-specific B3-domain transcription factor family have important and varied functions, especially with respect to vegetative and reproductive growth. Although B3 genes have been studied in many other plants, there is limited information on the genomic organization and expression of B3 genes in grapevine (Vitis vinifera L.). In this study, we identified 50 B3 genes in the grapevine genome and analyzed these genes in terms of chromosomal location and syntenic relationships, intron-exon organization, and promoter cis-element content. We also analyzed the presumed proteins in terms of domain structure and phylogenetic relationships. Based on the results, we classified these genes into five subfamilies. The syntenic relationships suggest that approximately half of the genes resulted from genome duplication, contributing to the expansion of the B3 family in grapevine. The analysis of cis-element composition suggested that most of these genes may function in response to hormones, light, and stress. We also analyzed expression of members of the B3 family in various structures of grapevine plants, including the seed during seed development. Many B3 genes were expressed preferentially in one or more structures of the developed plant, suggesting specific roles in growth and development. Furthermore, several of the genes were expressed differentially in early developing seeds from representative seeded and seedless cultivars, suggesting a role in seed development or abortion. The results of this study provide a foundation for functional analysis of B3 genes and new resources for future molecular breeding of grapevine.


Assuntos
Proteínas de Plantas/genética , Sementes/metabolismo , Fatores de Transcrição/genética , Vitis/genética , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Genômica , Germinação/genética , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Elementos Reguladores de Transcrição , Sintenia/genética , Fatores de Transcrição/metabolismo , Vitis/embriologia , Vitis/metabolismo
9.
PLoS Genet ; 15(9): e1008371, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527906

RESUMO

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.


Assuntos
Duplicação Cromossômica/genética , Segregação de Cromossomos/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico/fisiologia , Interferência de RNA , Proteínas de Ligação a RNA/genética , Elementos Reguladores de Transcrição/genética , Fuso Acromático/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/genética
10.
PLoS Biol ; 17(9): e3000378, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31479438

RESUMO

During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle-independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.


Assuntos
Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Metamorfose Biológica , Asas de Animais/crescimento & desenvolvimento , Animais , Drosophila melanogaster , Regulação da Expressão Gênica no Desenvolvimento , Elementos Reguladores de Transcrição , Asas de Animais/ultraestrutura
11.
Nat Commun ; 10(1): 4063, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492858

RESUMO

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.


Assuntos
Sistemas CRISPR-Cas , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , RNA Guia/genética , Elementos Reguladores de Transcrição/genética , Biologia Computacional/métodos , Epigênese Genética/genética , Epigenômica/métodos , Edição de Genes/métodos , Células HEK293 , Humanos , Células K562
12.
Nat Commun ; 10(1): 3583, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395865

RESUMO

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.


Assuntos
Biologia Computacional/métodos , Doença/genética , Mutagênese , Elementos Reguladores de Transcrição/genética , Linhagem Celular , Clonagem Molecular , Genoma Humano/genética , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo Único
13.
Genes Dev ; 33(17-18): 1159-1174, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371436

RESUMO

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.


Assuntos
Nucleossomos/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nuclease do Micrococo/metabolismo
14.
Nat Commun ; 10(1): 3597, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399572

RESUMO

Hox proteins belong to a family of transcription factors with similar DNA binding specificities that control animal differentiation along the antero-posterior body axis. Hox proteins are expressed in partially overlapping regions where each one is responsible for the formation of particular organs and structures through the regulation of specific direct downstream targets. Thus, explaining how each Hox protein can selectively control its direct targets from those of another Hox protein is fundamental to understand animal development. Here we analyse a cis regulatory module directly regulated by seven different Drosophila Hox proteins and uncover how different Hox class proteins differentially control its expression. We find that regulation by one or another Hox protein depends on the combination of three modes: Hox-cofactor dependent DNA-binding specificity; Hox-monomer binding sites; and interaction with positive and negative Hox-collaborator proteins. Additionally, we find that similar regulation can be achieved by Amphioxus orthologs, suggesting these three mechanisms are conserved from insects to chordates.


Assuntos
Proteínas de Drosophila/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Drosophila/embriologia , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/genética , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox , Genes de Insetos , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Elementos Reguladores de Transcrição/fisiologia , Fatores de Transcrição/genética
15.
BMC Genomics ; 20(1): 573, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296162

RESUMO

BACKGROUND: The genome of Banana bunchy top virus (BBTV) consists of at least six circular, single-stranded DNA components of ~ 1 kb in length. Some BBTV isolates may also carry satellite DNA molecules that are not essential for BBTV infection. The relation between multipartite DNA virus replication and their transcriptional levels and the underlying mechanism remain unclear. RESULTS: To understand the coordinated replication and transcription of the multiple genomic components, the absolute amounts of each BBTV DNA component were measured by real-time PCR (qPCR), and their transcriptional levels were determined by RNAseq and reverse transcription-qPCR (qRT-PCR). Significant differences were found in the absolute amounts of individual BBTV genomic components. Transcriptional levels of each BBTV genomic component obtained from the RNAseq data matched closely to those obtained from qRT-PCR, but did not correspond to the absolute amount of each DNA component. The ratio of transcript over DNA copies ranged from 46.21 to 1059.44%, which was possibly regulated by the promoter region in the intergenic region of each component. To further determine this speculation, the promoter region of the DNA-S, -M or -N was constructed to the upstream of green fluorescent protein (GFP) gene for transient expression by agrobacterium-mediated transformation method. The qRT-PCR showed the highest transcriptional activity was promoted by DNA-N promoter, about 386.58% activity comparing with CaMV 35S promoter. Confocal microscopy observation showed that the intensity of green fluorescence was corresponding to that of qRT-PCR. CONCLUSIONS: Our data clearly showed that BBTV was able to control the transcriptional level of each DNA component independently by through the promoter sequences in the intergenic region. Moreover, a cis-acting element from DNA-N component had a high transcriptional activity.


Assuntos
Babuvirus/genética , Genômica , Elementos Reguladores de Transcrição/genética , Transcrição Genética , Genoma Viral/genética , RNA Mensageiro/genética , Análise de Sequência de RNA
16.
Biochim Biophys Acta Gene Regul Mech ; 1862(11-12): 194392, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31276857

RESUMO

The generation of protein coding mRNAs from pre-mRNA is a fundamental biological process that is required for gene expression. Alternative pre-mRNA splicing is responsible for much of the transcriptomic and proteomic diversity observed in higher order eukaryotes. Aberrations that disrupt regular alternative splicing patterns are known to cause human diseases, including various cancers. Alternative splicing is a combinatorial process, meaning many factors affect which two splice sites are ligated together. The features that dictate exon inclusion are comprised of splice site strength, intron-exon architecture, RNA secondary structure, splicing regulatory elements, promoter use and transcription speed by RNA polymerase and the presence of post-transcriptional nucleotide modifications. A comprehensive view of all of the factors that influence alternative splicing decisions is necessary to predict splicing outcomes and to understand the molecular basis of disease. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.


Assuntos
Processamento Alternativo , RNA Mensageiro/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Sítios de Splice de RNA , RNA Mensageiro/química , Elementos Reguladores de Transcrição
17.
Nucleic Acids Res ; 47(14): 7235-7246, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31265076

RESUMO

Despite large experimental and computational efforts aiming to dissect the mechanisms underlying disease risk, mapping cis-regulatory elements to target genes remains a challenge. Here, we introduce a matrix factorization framework to integrate physical and functional interaction data of genomic segments. The framework was used to predict a regulatory network of chromatin interaction edges linking more than 20 000 promoters and 1.8 million enhancers across 127 human reference epigenomes, including edges that are present in any of the input datasets. Our network integrates functional evidence of correlated activity patterns from epigenomic data and physical evidence of chromatin interactions. An important contribution of this work is the representation of heterogeneous data with different qualities as networks. We show that the unbiased integration of independent data sources suggestive of regulatory interactions produces meaningful associations supported by existing functional and physical evidence, correlating with expected independent biological features.


Assuntos
Algoritmos , Biologia Computacional/métodos , Epigenômica/métodos , Redes Reguladoras de Genes , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genética , Cromatina/genética , Cromatina/metabolismo , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Humanos , Células K562 , Polimorfismo de Nucleotídeo Único , Mapeamento de Interação de Proteínas/métodos , Reprodutibilidade dos Testes
18.
Genome Biol ; 20(1): 139, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307500

RESUMO

BACKGROUND: Bread wheat is an allohexaploid species with a 16-Gb genome that has large intergenic regions, which presents a big challenge for pinpointing regulatory elements and further revealing the transcriptional regulatory mechanisms. Chromatin profiling to characterize the combinatorial patterns of chromatin signatures is a powerful means to detect functional elements and clarify regulatory activities in human studies. RESULTS: In the present study, through comprehensive analyses of the open chromatin, DNA methylome, seven major chromatin marks, and transcriptomic data generated for seedlings of allohexaploid wheat, we detected distinct chromatin architectural features surrounding various functional elements, including genes, promoters, enhancer-like elements, and transposons. Thousands of new genic regions and cis-regulatory elements are identified based on the combinatorial pattern of chromatin features. Roughly 1.5% of the genome encodes a subset of active regulatory elements, including promoters and enhancer-like elements, which are characterized by a high degree of chromatin openness and histone acetylation, an abundance of CpG islands, and low DNA methylation levels. A comparison across sub-genomes reveals that evolutionary selection on gene regulation is targeted at the sequence and chromatin feature levels. The divergent enrichment of cis-elements between enhancer-like sequences and promoters implies these functional elements are targeted by different transcription factors. CONCLUSIONS: We herein present a systematic epigenomic map for the annotation of cis-regulatory elements in the bread wheat genome, which provides new insights into the connections between chromatin modifications and cis-regulatory activities in allohexaploid wheat.


Assuntos
Montagem e Desmontagem da Cromatina , Metilação de DNA , Código das Histonas , Elementos Reguladores de Transcrição , Triticum/genética , Evolução Biológica , Epigenômica , Genoma de Planta , Plântula/metabolismo , Triticum/metabolismo
19.
Genome Biol ; 20(1): 133, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287004

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells. RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA. CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.


Assuntos
Doença da Artéria Coronariana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Sistemas CRISPR-Cas , Células Endoteliais/metabolismo , Epigenômica , Estudo de Associação Genômica Ampla , Humanos , Elementos Reguladores de Transcrição , Transcriptoma
20.
Nat Commun ; 10(1): 2880, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253799

RESUMO

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.


Assuntos
Aprendizado de Máquina , Regiões Promotoras Genéticas , Software , Neoplasias da Mama , Linhagem Celular Tumoral , Separação Celular/métodos , Feminino , Regulação da Expressão Gênica , Biblioteca Gênica , Glioblastoma , Humanos , Células-Tronco Pluripotentes Induzidas , Lentivirus , Células-Tronco Neoplásicas , Organoides , Elementos Reguladores de Transcrição
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