Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 171.303
Filtrar
1.
Nature ; 596(7871): 211-220, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34381231

RESUMO

Deciphering the principles and mechanisms by which gene activity orchestrates complex cellular arrangements in multicellular organisms has far-reaching implications for research in the life sciences. Recent technological advances in next-generation sequencing- and imaging-based approaches have established the power of spatial transcriptomics to measure expression levels of all or most genes systematically throughout tissue space, and have been adopted to generate biological insights in neuroscience, development and plant biology as well as to investigate a range of disease contexts, including cancer. Similar to datasets made possible by genomic sequencing and population health surveys, the large-scale atlases generated by this technology lend themselves to exploratory data analysis for hypothesis generation. Here we review spatial transcriptomic technologies and describe the repertoire of operations available for paths of analysis of the resulting data. Spatial transcriptomics can also be deployed for hypothesis testing using experimental designs that compare time points or conditions-including genetic or environmental perturbations. Finally, spatial transcriptomic data are naturally amenable to integration with other data modalities, providing an expandable framework for insight into tissue organization.


Assuntos
Perfilação da Expressão Gênica/métodos , Especificidade de Órgãos/genética , Transcriptoma , Animais , Análise de Dados , Doença/genética , Humanos , Transcrição Genética/genética
2.
Viruses ; 13(7)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34372533

RESUMO

Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.


Assuntos
Vírus da Hepatite B/genética , Hepatite B/prevenção & controle , Transcrição Genética/genética , Antivirais/farmacologia , DNA Circular/metabolismo , DNA Viral/genética , Hepatite B/tratamento farmacológico , Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Fígado/virologia , RNA Interferente Pequeno/metabolismo , Transcrição Genética/fisiologia , Integração Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445775

RESUMO

The onset of an early development is, in mammals, characterized by profound changes of multiple aspects of cellular morphology and behavior. These are including, but not limited to, fertilization and the merging of parental genomes with a subsequent transition from the meiotic into the mitotic cycle, followed by global changes of chromatin epigenetic modifications, a gradual decrease in cell size and the initiation of gene expression from the newly formed embryonic genome. Some of these important, and sometimes also dramatic, changes are executed within the period during which the gene transcription is globally silenced or not progressed, and the regulation of most cellular activities, including those mentioned above, relies on controlled translation. It is known that the blastomeres within an early embryo are prone to chromosome segregation errors, which might, when affecting a significant proportion of a cell within the embryo, compromise its further development. In this review, we discuss how the absence of transcription affects the transition from the oocyte to the embryo and what impact global transcriptional silencing might have on the basic cell cycle and chromosome segregation controlling mechanisms.


Assuntos
Ciclo Celular/genética , Cromatina/genética , Segregação de Cromossomos/genética , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/genética , Inativação Gênica/fisiologia , Transcrição Genética/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos
4.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445249

RESUMO

The SKP1, CUL1, F-box protein (SCF) complex encompasses a group of 69 SCF E3 ubiquitin ligase complexes that primarily modify protein substrates with poly-ubiquitin chains to target them for proteasomal degradation. These SCF complexes are distinguishable by variable F-box proteins, which determine substrate specificity. Although the function(s) of each individual SCF complex remain largely unknown, those that have been characterized regulate a wide array of cellular processes, including gene transcription and the cell cycle. In this regard, the SCF complex regulates transcription factors that modulate cell signaling and ensures timely degradation of primary cell cycle regulators for accurate replication and segregation of genetic material. SCF complex members are aberrantly expressed in a myriad of cancer types, with altered expression or function of the invariable core SCF components expected to have a greater impact on cancer pathogenesis than that of the F-box proteins. Accordingly, this review describes the normal roles that various SCF complexes have in maintaining genome stability before discussing the impact that aberrant SCF complex expression and/or function have on cancer pathogenesis. Further characterization of the SCF complex functions is essential to identify and develop therapeutic approaches to exploit aberrant SCF complex expression and function.


Assuntos
Cromossomos Humanos , Instabilidade Genômica , Proteínas de Neoplasias , Neoplasias , Proteínas Ligases SKP Culina F-Box , Transcrição Genética , Animais , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445254

RESUMO

Nitrogen forms (nitrate (NO3-) or ammonium (NH4+)) are vital to plant growth and metabolism. In stevia (Stevia rebaudiana), it is important to assess whether nitrogen forms can influence the synthesis of the high-value terpene metabolites-steviol glycosides (SGs), together with the underlying mechanisms. Field and pot experiments were performed where stevia plants were fertilized with either NO3- or NH4+ nutrition to the same level of nitrogen. Physiological measurements suggested that nitrogen forms had no significant impact on biomass and the total nitrogen content of stevia leaves, but NO3--enhanced leaf SGs contents. Transcriptomic analysis identified 397 genes that were differentially expressed (DEGs) between NO3- and NH4+ treatments. Assessment of the DEGs highlighted the responses in secondary metabolism, particularly in terpenoid metabolism, to nitrogen forms. Further examinations of the expression patterns of SGs synthesis-related genes and potential transcription factors suggested that GGPPS and CPS genes, as well as the WRKY and MYB transcription factors, could be driving N form-regulated SG synthesis. We concluded that NO3-, rather than NH4+, can promote leaf SG synthesis via the NO3--MYB/WRKY-GGPPS/CPS module. Our study suggests that insights into the molecular mechanism of how SG synthesis can be affected by nitrogen forms.


Assuntos
Amônia/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucosídeos/biossíntese , Nitratos/metabolismo , Stevia/metabolismo , Transcrição Genética/efeitos dos fármacos , Diterpenos do Tipo Caurano , Perfilação da Expressão Gênica , Glucosídeos/genética , Nitratos/farmacologia , Stevia/genética
6.
Nucleic Acids Res ; 49(15): 8796-8810, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379778

RESUMO

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.


Assuntos
Vírus da Influenza A/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico , Cães , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino , Mutação , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Proteínas Virais/genética
7.
Nat Immunol ; 22(9): 1152-1162, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34385712

RESUMO

The transcription factor TCF-1 is essential for the development and function of regulatory T (Treg) cells; however, its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-sequencing analysis identified effector memory T cells and central memory Treg cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg cell transcriptional signature, but promoted alternative signaling pathways whereby Treg cells became activated and gained gut-homing properties and characteristics of the TH17 subset of helper T cells. TCF-1-deficient Treg cells strongly suppressed T cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of patients with colorectal cancer showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T cell cytotoxicity, and can determine colorectal cancer outcome.


Assuntos
Neoplasias do Colo/patologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/imunologia , Animais , Proliferação de Células/fisiologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Fator 1-alfa Nuclear de Hepatócito/genética , Memória Imunológica/imunologia , Inflamação/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Transcrição Genética/genética , Proteínas Supressoras de Tumor/metabolismo
8.
FASEB J ; 35(9): e21667, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34405442

RESUMO

Long noncoding RNAs (lncRNAs) are central regulators of the inflammatory response and play an important role in inflammatory diseases. PINT has been reported to be involved in embryonic development and tumorigenesis. However, the potential functions of PINT in the innate immune system are largely unknown. Here, we revealed the transcriptional regulation of inflammatory genes by PINT, whose expression is primarily dependent on the NF-κB signaling pathway in human and mouse macrophage and intestinal epithelial cell lines. Functionally, PINT selectively regulates the expression of TNF-α in basal and LPS-stimulated cells. Mechanistically, PINT acts as a modular scaffold of p65 and EZH2 to coordinate their localization and specify their binding to the target genes. Further, a high expression level of PINT was detected in intestinal mucosal tissues from patients with ulcerative colitis (UC). Together, these findings demonstrate that PINT acts as an activator of inflammatory responses, highlighting the importance of this lncRNA as a potential therapeutic target in infectious diseases and inflammatory diseases.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Fator de Transcrição RelA/metabolismo , Transcrição Genética , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Citocinas/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transcrição Genética/genética
9.
Science ; 373(6556): 760-767, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385390

RESUMO

The origin of human metaplastic states and their propensity for cancer is poorly understood. Barrett's esophagus is a common metaplastic condition that increases the risk for esophageal adenocarcinoma, and its cellular origin is enigmatic. To address this, we harvested tissues spanning the gastroesophageal junction from healthy and diseased donors, including isolation of esophageal submucosal glands. A combination of single-cell transcriptomic profiling, in silico lineage tracing from methylation, open chromatin and somatic mutation analyses, and functional studies in organoid models showed that Barrett's esophagus originates from gastric cardia through c-MYC and HNF4A-driven transcriptional programs. Furthermore, our data indicate that esophageal adenocarcinoma likely arises from undifferentiated Barrett's esophagus cell types even in the absence of a pathologically identifiable metaplastic precursor, illuminating early detection strategies.


Assuntos
Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Cárdia/citologia , Neoplasias Esofágicas/patologia , Esôfago/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Cárdia/química , Diferenciação Celular , Linhagem da Célula , Transformação Celular Neoplásica , Epigênese Genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Glândulas Exócrinas/química , Glândulas Exócrinas/citologia , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Queratina-7/análise , Metaplasia , Fenótipo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA-Seq , Análise de Célula Única , Transcrição Genética , Transcriptoma
10.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
11.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360611

RESUMO

Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many cases, the required speed and plasticity of the response are provided by RNA-dependent regulatory mechanisms. This is possible due to the very high dynamics and flexibility of an RNA structure, which provide the necessary sensitivity and specificity for efficient sensing and transduction of environmental signals. In this review, we will discuss the current knowledge about known bacterial regulatory mechanisms which rely on RNA structure. To better understand the structure-driven modulation of gene expression, we describe the basic theory on RNA structure folding and dynamics. Next, we present examples of multiple mechanisms employed by RNA regulators in the control of bacterial transcription and translation.


Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Bacteriano/química , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Proteínas de Bactérias/genética , Transcrição Genética
12.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360615

RESUMO

In contrast to Bacillus subtilis, Streptomyces coelicolor A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from S. coelicolor A3(2). Here, we used this system to identify 14 promoters recognized by SigB. The promoters were verified in vivo in S. coelicolor A3(2) under osmotic stress conditions in sigB and sigH operon mutants, indicating some cross-recognition of these promoters by these two SigB homologues. This two-plasmid system was used to examine the recognition of all identified SigB-, SigF-, SigG-, and SigH-dependent promoters with all nine SigB homologues. The results confirmed this cross-recognition. Almost all 24 investigated promoters were recognized by two or more SigB homologues and data suggested some distinguishing groups of promoters recognized by these sigma factors. However, analysis of the promoters did not reveal any specific sequence characteristics for these recognition groups. All promoters showed high similarity in the -35 and -10 regions. Immunoblot analysis revealed the presence of SigB under osmotic stress conditions and SigH during morphological differentiation. Together with the phenotypic analysis of sigB and sigH operon mutants in S. coelicolor A3(2), the results suggest a dominant role for SigB in the osmotic stress response and a dual role for SigH in the osmotic stress response and morphological differentiation. These data suggest a complex regulation of the osmotic stress response in relation to morphological differentiation in S. coelicolor A3(2).


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras Genéticas , Fator sigma/genética , Streptomyces coelicolor/genética , Transcrição Genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Fator sigma/metabolismo , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/metabolismo
13.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360962

RESUMO

Somatic cell nuclear transfer (SCNT) technology can reprogram terminally differentiated cell nuclei into a totipotent state. However, the underlying molecular barriers of SCNT embryo development remain incompletely elucidated. Here, we observed that transcription-related pathways were incompletely activated in nuclear transfer arrest (NTA) embryos compared to normal SCNT embryos and in vivo fertilized (WT) embryos, which hinders the development of SCNT embryos. We further revealed the transcription pathway associated gene regulatory networks (GRNs) and found the aberrant transcription pathways can lead to the massive dysregulation of genes in NTA embryos. The predicted target genes of transcription pathways contain a series of crucial factors in WT embryos, which play an important role in catabolic process, pluripotency regulation, epigenetic modification and signal transduction. In NTA embryos, however, these genes were varying degrees of inhibition and show a defect in synergy. Overall, our research found that the incomplete activation of transcription pathways is another potential molecular barrier for SCNT embryos besides the incomplete reprogramming of epigenetic modifications, broadening the understanding of molecular mechanism of SCNT embryonic development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Técnicas de Transferência Nuclear/efeitos adversos , Transcriptoma , Animais , Blastocisto/metabolismo , Camundongos , RNA-Seq , Análise de Célula Única , Transcrição Genética
14.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Genética
15.
Nat Commun ; 12(1): 4919, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389725

RESUMO

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Genética/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismo
16.
Nat Commun ; 12(1): 4929, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389727

RESUMO

Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.


Assuntos
Escherichia coli/genética , Glicólise/genética , Engenharia Metabólica/métodos , Metabolômica/métodos , Proteômica/métodos , Transcrição Genética , Algoritmos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Carotenoides/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Methods Mol Biol ; 2351: 25-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382182

RESUMO

Post-transcriptional processing strongly affects the stability and the relative quantification of RNA molecules, so that steady-state levels of mature RNA, such as mRNAs, rarely reflect accurately the rate of in situ transcription in nuclei by RNA polymerases (RNAPs). The "Global Run-on Sequencing (GRO-Seq)" method, developed in 2008, combines the nuclear run-on assay with next-generation deep sequencing to detect nascent RNA levels to annotate the positions, the relative levels and the orientation of transcriptionally engaged RNA polymerase II (RNAPII) molecules genome-wide. Thus, GRO-Seq is a powerful method to infer mechanistic insights into the multiple levels of transcriptional regulation such as promoter-proximal pausing of RNAP, bidirectional transcription, and enhancer activity. Here, we describe a protocol for mammalian cells that can reliably detect low abundant nascent RNA from both coding and noncoding genomic regions. This protocol can easily be adapted for most mammalian cells to define the transcriptionally active regions of the genome and to measure dynamic transcriptional responses with high sensitivity upon external stimuli.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Polimerase II/metabolismo , Análise de Sequência de RNA/métodos , Transcrição Genética , Elementos Facilitadores Genéticos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Controle de Qualidade , RNA/genética , RNA/isolamento & purificação , RNA não Traduzido/genética
18.
Methods Mol Biol ; 2351: 41-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382183

RESUMO

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.


Assuntos
Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Transcrição Genética , Animais , Células Cultivadas , Cromatina/genética , Biologia Computacional/métodos , DNA , Biblioteca Gênica , Humanos , Reação em Cadeia da Polimerase , RNA , RNA Polimerase II/metabolismo , Software
19.
Methods Mol Biol ; 2351: 3-22, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382181

RESUMO

Knowledge in gene transcription and chromatin regulation has been intensely studied for decades, but thanks to next-generation sequencing (NGS) techniques there has been a major leap forward in the last few years. Historically, identification of specific enhancer elements has led to the identification of master transcription factors (TFs) in the 1990s. Genetic and biochemical experiments have identified the key regulators controlling RNA polymerase II (RNAPII) transcription and structurally analyses have elucidated detailed mechanisms. NGS and the development of chromatin immunoprecipitation (ChIP) have accelerated the gain of knowledge in the recent years. By now, we have a dazzling wealth of techniques that are currently used to put gene expression into a genome-wide context. This book is an attempt to assemble useful protocols for many researchers within and nearby research areas. In general, these innovative techniques focus on enhancer and promoter studies. The techniques should also be of interest for related fields such as DNA repair and replication.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Genética , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Humanos , Ligação Proteica , RNA Polimerase II/metabolismo
20.
Methods Mol Biol ; 2351: 201-210, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382191

RESUMO

Regulation of gene expression is a key feature for higher eukaryotes and how chromatin topology relates to gene activation is an intense area of research. Enhancer-promoter interactions are believed to mediate activation of target genes. Bidirectional transcription represents one hallmark of active enhancers that can be measured using transcriptome technologies such as Cap analysis of gene expression (CAGE). Recently, we have developed RNA and DNA interacting complexes ligated and sequenced (RADICL-Seq) a novel methodology to map genome-wide RNA-chromatin interactions in intact nuclei. Here, we describe how CAGE and RADICL-Seq data can be used to characterize enhancer elements and identify their target genes.


Assuntos
Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Capuzes de RNA , Algoritmos , Cromatina/genética , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Sítio de Iniciação de Transcrição , Transcrição Genética , Ativação Transcricional , Transcriptoma
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...