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1.
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
2.
Nat Commun ; 11(1): 3340, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620845

RESUMO

GWAS cannot identify functional SNPs (fSNP) from disease-associated SNPs in linkage disequilibrium (LD). Here, we report developing three sequential methodologies including Reel-seq (Regulatory element-sequencing) to identify fSNPs in a high-throughput fashion, SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify fSNP-bound proteins and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to detect allele-specific protein:fSNP binding. We first apply Reel-seq to screen a library containing 4316 breast cancer-associated SNPs and identify 521 candidate fSNPs. As proof of principle, we verify candidate fSNPs on three well-characterized loci: FGFR2, MAP3K1 and BABAM1. Next, using SDCP-MS and AIDP-Wb, we rapidly identify multiple regulatory factors that specifically bind in an allele-imbalanced manner to the fSNPs on the FGFR2 locus. We finally demonstrate that the factors identified by SDCP-MS can regulate risk gene expression. These data suggest that the sequential application of Reel-seq, SDCP-MS, and AIDP-Wb can greatly help to translate large sets of GWAS data into biologically relevant information.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Desequilíbrio de Ligação , MAP Quinase Quinase Quinase 1/genética , Células MCF-7 , Espectrometria de Massas/métodos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Sequências Reguladoras de Ácido Nucleico/genética
3.
Proc Natl Acad Sci U S A ; 117(29): 17130-17134, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636262

RESUMO

Supergenes underlie striking polymorphisms in nature, yet the evolutionary mechanisms by which they arise and persist remain enigmatic. These clusters of linked loci can spread in populations because they captured coadapted alleles or by selfishly distorting the laws of Mendelian inheritance. Here, we show that the supergene haplotype associated with multiple-queen colonies in Alpine silver ants is a maternal effect killer. All eggs from heterozygous queens failed to hatch when they did not inherit this haplotype. Hence, the haplotype specific to multiple-queen colonies is a selfish genetic element that enhances its own transmission by causing developmental arrest of progeny that do not carry it. At the population level, such transmission ratio distortion favors the spread of multiple-queen colonies, to the detriment of the alternative haplotype associated with single-queen colonies. Hence, selfish gene drive by one haplotype will impact the evolutionary dynamics of alternative forms of colony social organization. This killer hidden in a social supergene shows that large nonrecombining genomic regions are prone to cause multifarious effects across levels of biological organization.


Assuntos
Formigas/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes de Insetos/genética , Herança Materna/genética , Comportamento Social , Animais , Formigas/crescimento & desenvolvimento , Formigas/fisiologia , Evolução Molecular , Feminino , Haplótipos/genética , Masculino , Meiose/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sequências Repetitivas de Ácido Nucleico/genética
4.
Nature ; 584(7820): 244-251, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728217

RESUMO

DNase I hypersensitive sites (DHSs) are generic markers of regulatory DNA1-5 and contain genetic variations associated with diseases and phenotypic traits6-8. We created high-resolution maps of DHSs from 733 human biosamples encompassing 438 cell and tissue types and states, and integrated these to delineate and numerically index approximately 3.6 million DHSs within the human genome sequence, providing a common coordinate system for regulatory DNA. Here we show that these maps highly resolve the cis-regulatory compartment of the human genome, which encodes unexpectedly diverse cell- and tissue-selective regulatory programs at very high density. These programs can be captured comprehensively by a simple vocabulary that enables the assignment to each DHS of a regulatory barcode that encapsulates its tissue manifestations, and global annotation of protein-coding and non-coding RNA genes in a manner orthogonal to gene expression. Finally, we show that sharply resolved DHSs markedly enhance the genetic association and heritability signals of diseases and traits. Rather than being confined to a small number of distal elements or promoters, we find that genetic signals converge on congruently regulated sets of DHSs that decorate entire gene bodies. Together, our results create a universal, extensible coordinate system and vocabulary for human regulatory DNA marked by DHSs, and provide a new global perspective on the architecture of human gene regulation.


Assuntos
Cromatina/genética , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Anotação de Sequência Molecular , Cromatina/química , Cromatina/metabolismo , DNA/química , DNA/genética , Regulação da Expressão Gênica , Genes/genética , Genoma Humano/genética , Humanos , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética
5.
Nat Protoc ; 15(8): 2387-2412, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641802

RESUMO

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.


Assuntos
Lentivirus/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA/métodos , Fluxo de Trabalho , Sequência de Bases
6.
PLoS One ; 15(7): e0235530, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614871

RESUMO

BACKGROUND: Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity of renin transcription in its native genomic context. METHODS: With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models. RESULTS: SAM complexes induced activation of renin in our cells after renin specific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data. CONCLUSION: By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Sequências Reguladoras de Ácido Nucleico/genética , Genes Reporter , Células HEK293 , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Regiões Promotoras Genéticas , RNA Guia/metabolismo , Renina/genética
7.
Nat Commun ; 11(1): 3551, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669542

RESUMO

Predicting effects of gene regulatory elements (GREs) is a longstanding challenge in biology. Machine learning may address this, but requires large datasets linking GREs to their quantitative function. However, experimental methods to generate such datasets are either application-specific or technically complex and error-prone. Here, we introduce DNA-based phenotypic recording as a widely applicable, practicable approach to generate large-scale sequence-function datasets. We use a site-specific recombinase to directly record a GRE's effect in DNA, enabling readout of both sequence and quantitative function for extremely large GRE-sets via next-generation sequencing. We record translation kinetics of over 300,000 bacterial ribosome binding sites (RBSs) in >2.7 million sequence-function pairs in a single experiment. Further, we introduce a deep learning approach employing ensembling and uncertainty modelling that predicts RBS function with high accuracy, outperforming state-of-the-art methods. DNA-based phenotypic recording combined with deep learning represents a major advance in our ability to predict function from genetic sequence.


Assuntos
Biologia Computacional/métodos , Aprendizado Profundo , Anotação de Sequência Molecular/métodos , Fenótipo , Análise de Sequência de DNA/métodos , Sítios de Ligação/genética , Conjuntos de Dados como Assunto , Escherichia coli/genética , Técnicas de Inativação de Genes , Genoma Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Sequências Reguladoras de Ácido Nucleico/genética , Ribossomos/metabolismo
8.
Nature ; 583(7814): 90-95, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499645

RESUMO

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies1-3. Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed4) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of-and interplay between-novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.


Assuntos
Doenças da Imunodeficiência Primária/genética , Sequenciamento Completo do Genoma , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Teorema de Bayes , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Doenças da Imunodeficiência Primária/diagnóstico , Doenças da Imunodeficiência Primária/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Fatores de Transcrição/genética
9.
Nat Commun ; 11(1): 2680, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471981

RESUMO

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Desmetilação do DNA , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Epigênese Genética/genética , Genoma/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/genética
10.
Mol Genet Genomics ; 295(4): 1013-1026, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32363570

RESUMO

Obesity, a risk factor for multiple diseases (e.g. diabetes, hypertension, cancers) originates through complex interactions between genes and prevailing environment (food habit and lifestyle) that varies across populations. Indians exhibit a unique obesity phenotype with high abdominal adiposity for a given body weight compared to matched white populations suggesting presence of population-specific genetic and environmental factors influencing obesity. However, Indian population-specific genetic contributors for obesity have not been explored yet. Therefore, to identify potential genetic contributors, we performed a two-staged genome-wide association study (GWAS) for body mass index (BMI), a common measure to evaluate obesity in 5973 Indian adults and the lead findings were further replicated in 1286 Indian adolescents. Our study revealed novel association of variants-rs6913677 in BAI3 gene (p = 1.08 × 10-8) and rs2078267 in SLC22A11 gene (p = 4.62 × 10-8) at GWAS significance, and of rs8100011 in ZNF45 gene (p = 1.04 × 10-7) with near GWAS significance. As genetic loci may dictate the phenotype through modulation of epigenetic processes, we overlapped genetic data of identified signals with their DNA methylation patterns in 236 Indian individuals and performed methylation quantitative trait loci (meth-QTL) analysis. Further, functional roles of discovered variants and underlying genes were speculated using publicly available gene regulatory databases (ENCODE, JASPAR, GeneHancer, GTEx). The identified variants in BAI3 and SLC22A11 genes were found to dictate methylation patterns at unique CpGs harboring critical cis-regulatory elements. Further, BAI3, SLC22A11 and ZNF45 variants were located in repressive chromatin, active enhancer, and active chromatin regions, respectively, in human subcutaneous adipose tissue in ENCODE database. Additionally, these genomic regions represented potential binding sites for key transcription factors implicated in obesity and/or metabolic disorders. Interestingly, GTEx portal identify rs8100011 as a robust cis-expression quantitative trait locus (cis-eQTL) in subcutaneous adipose tissue (p = 1.6 × 10-7), and ZNF45 gene expression in skeletal muscle of Indian subjects showed an inverse correlation with BMI indicating its possible role in obesity. In conclusion, our study discovered 3 novel population-specific functional genetic variants (rs6913677, rs2078267, rs8100011) in 2 novel (SLC22A11 and ZNF45) and 1 earlier reported gene (BAI3) for BMI in Indians. Our study decodes key genomic loci underlying obesity phenotype in Indians that may serve as prospective drug targets in future.


Assuntos
Estudo de Associação Genômica Ampla , Fatores de Transcrição Kruppel-Like/genética , Obesidade/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Índice de Massa Corporal , Metilação de DNA , Feminino , Interação Gene-Ambiente , Predisposição Genética para Doença , Humanos , Índios Norte-Americanos/genética , Masculino , Obesidade/patologia , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Adulto Jovem
11.
Gene ; 747: 144674, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32304781

RESUMO

Very long chain fatty acids (VLCFAs) that are structural components of cell membrane lipid, cuticular waxes and seed oil, play crucial roles in plant growth, development and stress response. Fatty acid elongases (FAEs) comprising KCS and ELO, are key enzymes for VLCFA biosynthesis in plants. Although reference genomes of Brassica napus and its parental speices both have been sequenced, whole-genome analysis of FAE gene family in these Brassica speices is not reported. Here, 58, 33 and 30 KCS genes were identified in B. napus, B. rapa and B. oleracea genomes, respectively, whereas 14, 6 and 8 members were obtained for ELO genes. These KCS genes were unevenly located in 37 chromosomes and 3 scaffolds of 3 Brassica species, while these ELO genes were mapped to 19 chromosomes. The KCS and ELO proteins were divided into 8 and 4 subclasses, respectively. Gene structure and protein motifs remained highly conserved in each KCS or ELO subclass. Most promoters of KCS and ELO genes harbored various plant growth-, phytohormone-, and stress response-related cis-acting elements. 20 SSR loci existed in the KCS and ELO genes/promoters. The whole-genome duplication and segmental duplication mainly contributed to expansion of KCS and ELO genes in these genomes. Transcriptome analysis showed that KCS and ELO genes in 3 Brassica species were expressed in various tissues/organs with different levels, whereas 1 BnELO gene and 6 BnKCS genes might be pathogen-responsive genes. The qRT-PCR assay showed that BnKCS22 and BnELO04 responded to various phytohormone treatments and abiotic stresses. This work lays the foundation for further function identification of KCS and ELO genes in B. napus and its progenitors.


Assuntos
Brassica napus/enzimologia , Brassica napus/genética , Elongases de Ácidos Graxos/genética , Genes de Plantas , Estudo de Associação Genômica Ampla , Família Multigênica , Brassica napus/efeitos dos fármacos , Cromossomos de Plantas/genética , Sequência Conservada/genética , Duplicação Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Loci Gênicos , Repetições de Microssatélites/genética , Motivos de Nucleotídeos , Filogenia , Reguladores de Crescimento de Planta/farmacologia , Sequências Reguladoras de Ácido Nucleico/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Sintenia/genética
12.
Mol Cell ; 78(2): 289-302.e6, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302541

RESUMO

Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor.


Assuntos
Proteínas de Ciclo Celular/genética , MicroRNAs/genética , RNA Polimerase III/genética , Fatores de Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Genética
13.
Proc Natl Acad Sci U S A ; 117(16): 9001-9012, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32265282

RESUMO

The interplay of transcription factors and cis-regulatory elements (CREs) orchestrates the dynamic and diverse genetic programs that assemble the human central nervous system (CNS) during development and maintain its function throughout life. Genetic variation within CREs plays a central role in phenotypic variation in complex traits including the risk of developing disease. We took advantage of the retina, a well-characterized region of the CNS known to be affected by pathogenic variants in CREs, to establish a roadmap for characterizing regulatory variation in the human CNS. This comprehensive analysis of tissue-specific regulatory elements, transcription factor binding, and gene expression programs in three regions of the human visual system (retina, macula, and retinal pigment epithelium/choroid) reveals features of regulatory element evolution that shape tissue-specific gene expression programs and defines regulatory elements with the potential to contribute to Mendelian and complex disorders of human vision.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Sequências Reguladoras de Ácido Nucleico/genética , Retina/patologia , Doenças Retinianas/genética , Adulto , Animais , Análise Mutacional de DNA , Epigenômica , Feminino , Variação Genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , RNA-Seq , Retina/crescimento & desenvolvimento , Doenças Retinianas/patologia , Especificidade da Espécie
14.
Nat Genet ; 52(3): 273-282, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32139906

RESUMO

Mutations in enzymes that modify histone H3 at lysine 4 (H3K4) or lysine 36 (H3K36) have been linked to human disease, yet the role of these residues in mammals is unclear. We mutated K4 or K36 to alanine in the histone variant H3.3 and showed that the K4A mutation in mouse embryonic stem cells (ESCs) impaired differentiation and induced widespread gene expression changes. K4A resulted in substantial H3.3 depletion, especially at ESC promoters; it was accompanied by reduced remodeler binding and increased RNA polymerase II (Pol II) activity. Regulatory regions depleted of H3.3K4A showed histone modification alterations and changes in enhancer activity that correlated with gene expression. In contrast, the K36A mutation did not alter H3.3 deposition and affected gene expression at the later stages of differentiation. Thus, H3K4 is required for nucleosome deposition, histone turnover and chromatin remodeler binding at regulatory regions, where tight regulation of Pol II activity is necessary for proper ESC differentiation.


Assuntos
Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Código das Histonas/genética , Histonas/genética , Lisina/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Alanina/metabolismo , Animais , Elementos Facilitadores Genéticos/genética , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Genética
15.
DNA Cell Biol ; 39(4): 563-571, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32073892

RESUMO

Growth differentiation factor (GDF) 9 gene is involved in regulating reproductive traits in animals, but little is known about the promoter, single-nucleotide polymorphisms (SNPs), transcription factor binding sites, and regulating mechanism of GDF9 gene. In this study, the SNPs in the GDF9 promoter region were explored and their transcription mechanisms in regulating GDF9 expression were analyzed. Ear tissues of 267 Hu ewes were collected, and genomic DNA was extracted. GDF9 promoter region was amplified by PCRs, and identified SNPs genotyped by sequencing. SPSS16.0 software was used to analyze the association between genotypes and litter sizes. Flow cytometry assay was used to detect cell apoptosis, and dual-luciferase reporter assay was used to discover the promoter activity. A length of 1789 bp promoter region of GDF9 in Hu sheep was obtained by PCR amplification, and luciferase activity assay showed that there was a negative regulatory element in the region within -725 to -309 bp and a positive regulatory element in the region within -309 to +43 bp. Three complete linkage SNPs at -534A/G, -407T/G, and -332C/T were detected, resulting in three genotypes (namely, AA, AB, and BB). The association analysis indicated that the AA genotype ewes had larger litter size at average parity than those with the BB genotype. The -534A/G mutation created a novel binding site for the octamer transcription factor 1 (OCT1), and the Annexin V FITC/PI flow cytometry assay showed OCT1 promoted cell apoptosis in sheep ovarian granulosa cells. Overexpression of OCT1 considerably inhibited the luciferase activity of both genotypes and the inhibition effect of pGL3-BB was higher than that of pGL3-AA. Three complete linkage SNPs of the GDF9 gene regulate the litter size in Hu sheep probably via inhibition of the promoter activity by binding with OCT1 at -534 GG genotype and forming a complex between OCT1 and CCAAT/enhancer-binding protein (C/EBP).


Assuntos
Fator 9 de Diferenciação de Crescimento/genética , Tamanho da Ninhada de Vivíparos/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Apoptose/fisiologia , Sítios de Ligação/genética , Células COS , Linhagem Celular , Chlorocebus aethiops , Feminino , Células da Granulosa/metabolismo , Fator 1 de Transcrição de Octâmero/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Sequências Reguladoras de Ácido Nucleico/genética , Ovinos
16.
PLoS One ; 15(2): e0228005, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32027681

RESUMO

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.


Assuntos
Calcitonina/genética , Carcinoma Neuroendócrino/terapia , Dependovirus/genética , Vetores Genéticos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Neoplasias da Glândula Tireoide/terapia , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Luciferases/metabolismo , Masculino , Camundongos SCID , Regiões Promotoras Genéticas , Transgenes , Ensaios Antitumorais Modelo de Xenoenxerto
17.
PLoS One ; 15(2): e0228845, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053662

RESUMO

Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Transporte Biológico , Regulação Fúngica da Expressão Gênica/genética , Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Quinases da Glicogênio Sintase/metabolismo , Quinases da Glicogênio Sintase/fisiologia , Oxigênio/metabolismo , Fosforilação , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Esteróis , Fatores de Transcrição/metabolismo , Ativação Transcricional
18.
BMC Evol Biol ; 20(1): 17, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005133

RESUMO

BACKGROUND: Drosophila subobscura exhibits a rich inversion polymorphism, with some adaptive inversions showing repeatable spatiotemporal patterns in frequencies related to temperature. Previous studies reported increased basal HSP70 protein levels in homokaryotypic strains for a warm-climate arrangement compared to a cold-climate one. These findings do not match the similar hsp70 genomic organization between arrangements, where gene expression levels are expected to be similar. In order to test this hypothesis and understand the molecular basis for hsp70 expression, we compared basal hsp70 mRNA levels in males and females, and analysed the 5' and 3' regulatory regions of hsp70 genes in warm- and cold-climate isochromosomal O3 + 4 + 7 and OST lines of D. subobscura. RESULTS: We observed comparable mRNA levels between the two arrangements and a sex-biased hsp70 gene expression. The number of heat-shock elements (HSEs) and GAGA sites on the promoters were identical amongst the OST and O3 + 4 + 7 lines analysed. This is also true for 3' AU-rich elements where most A and B copies of hsp70 have, respectively, two and one element in both arrangements. Beyond the regulatory elements, the only notable difference between both arrangements is the presence in 3' UTR of a 14 bp additional fragment after the stop codon in the hsp70A copy in five O3 + 4 + 7 lines, which was not found in any of the six OST lines. CONCLUSIONS: The equivalent hsp70 mRNA amounts in OST and O3 + 4 + 7 arrangements provide the first evidence of a parallelism between gene expression and genetic organization in D. subobscura lines having these arrangements. This is reinforced by the lack of important differential features in the number and structure of regulatory elements between both arrangements, despite the genetic differentiation observed when the complete 5' and 3' regulatory regions were considered. Therefore, the basal levels of hsp70 mRNA cannot account, in principle, for the adaptive variation of the two arrangements studied. Consequently, further studies are necessary to understand the intricate molecular mechanisms of hsp70 gene regulation in D. subobscura.


Assuntos
Adaptação Fisiológica/genética , Clima , Drosophila/genética , Regulação da Expressão Gênica , Rearranjo Gênico/genética , Genes de Insetos , Variação Genética , Proteínas de Choque Térmico HSP70/genética , Regiões 3' não Traduzidas/genética , Análise de Variância , Animais , Sequência de Bases , Sequência Conservada , Drosophila/fisiologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética
19.
BMC Evol Biol ; 20(1): 33, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32106815

RESUMO

BACKGROUND: Human chromosome 19 has many unique characteristics including gene density more than double the genome-wide average and 20 large tandemly clustered gene families. It also has the highest GC content of any chromosome, especially outside gene clusters. The high GC content and concomitant high content of hypermutable CpG sites raises the possibility chromosome 19 exhibits higher levels of nucleotide diversity both within and between species, and may possess greater variation in DNA methylation that regulates gene expression. RESULTS: We examined GC and CpG content of chromosome 19 orthologs across representatives of the primate order. In all 12 primate species with suitable genome assemblies, chromosome 19 orthologs have the highest GC content of any chromosome. CpG dinucleotides and CpG islands are also more prevalent in chromosome 19 orthologs than other chromosomes. GC and CpG content are generally higher outside the gene clusters. Intra-species variation based on SNPs in human common dbSNP, rhesus, crab eating macaque, baboon and marmoset datasets is most prevalent on chromosome 19 and its orthologs. Inter-species comparisons based on phyloP conservation show accelerated nucleotide evolution for chromosome 19 promoter flanking and enhancer regions. These same regulatory regions show the highest CpG density of any chromosome suggesting they possess considerable methylome regulatory potential. CONCLUSIONS: The pattern of high GC and CpG content in chromosome 19 orthologs, particularly outside gene clusters, is present from human to mouse lemur representing 74 million years of primate evolution. Much CpG variation exists both within and between primate species with a portion of this variation occurring in regulatory regions.


Assuntos
Cromossomos Humanos Par 19/genética , Sequência Conservada , Primatas/classificação , Primatas/genética , Animais , Composição de Bases , Sequência de Bases , Cromossomos/genética , Sequência Conservada/genética , Ilhas de CpG , Metilação de DNA , Fosfatos de Dinucleosídeos/genética , Genoma , Humanos , Lemur/classificação , Lemur/genética , Camundongos , Família Multigênica , Filogenia , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética
20.
RNA ; 26(3): 324-344, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31896558

RESUMO

Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and regulate mRNA poly(A) tail length to control protein production. However, the genome-wide relationship of poly(A) tail changes to mRNA translation during vertebrate oocyte maturation is not known. We used Tail-seq and polyribosome analysis to measure poly(A) tail and translational changes during oocyte maturation in Xenopus laevis We identified large-scale poly(A) and translational changes during oocyte maturation, with poly(A) tail length changes preceding translational changes. Proteins important for completion of the meiotic divisions and early development exhibited increased polyadenylation and translation during oocyte maturation. A family of U-rich sequence elements was enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. We propose that changes in mRNA polyadenylation are a conserved mechanism regulating protein expression during vertebrate oocyte maturation and that these changes are controlled by a spatial code of cis-acting sequence elements.


Assuntos
Oogênese/genética , Poliadenilação/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
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