RESUMO
Gene expression in Escherichia coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of ß and σ70 subunits to stabilize an open-promoter complex. CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis.
Assuntos
Proteínas de Escherichia coli , Fatores Genéricos de Transcrição , Escherichia coli/metabolismo , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Bactérias/metabolismoRESUMO
OBJECTIVE: Epigenetic abnormalities have a critical role in breast cancer by regulating gene expression; however, the intricate interrelationships and key roles of approximately 400 epigenetic regulators in breast cancer remain elusive. It is important to decipher the comprehensive epigenetic regulatory network in breast cancer cells to identify master epigenetic regulators and potential therapeutic targets. METHODS: We employed high-throughput sequencing-based high-throughput screening (HTS2) to effectively detect changes in the expression of 2,986 genes following the knockdown of 400 epigenetic regulators. Then, bioinformatics analysis tools were used for the resulting gene expression signatures to investigate the epigenetic regulations in breast cancer. RESULTS: Utilizing these gene expression signatures, we classified the epigenetic regulators into five distinct clusters, each characterized by specific functions. We discovered functional similarities between BAZ2B and SETMAR, as well as CLOCK and CBX3. Moreover, we observed that CLOCK functions in a manner opposite to that of HDAC8 in downstream gene regulation. Notably, we constructed an epigenetic regulatory network based on the gene expression signatures, which revealed 8 distinct modules and identified 10 master epigenetic regulators in breast cancer. CONCLUSIONS: Our work deciphered the extensive regulation among hundreds of epigenetic regulators. The identification of 10 master epigenetic regulators offers promising therapeutic targets for breast cancer treatment.
Assuntos
Neoplasias da Mama , Fatores Genéricos de Transcrição , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Redes Reguladoras de Genes , Regulação Neoplásica da Expressão Gênica , Biologia Computacional/métodos , Epigênese Genética/genética , Histona Desacetilases/genética , Proteínas Repressoras/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas que Contêm Bromodomínio , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismoRESUMO
Neurodegenerative disorders, such as Alzheimer's disease (AD) or Huntington's disease (HD), are linked to protein aggregate neurotoxicity. According to the "cholinergic hypothesis," loss of acetylcholine (ACh) signaling contributes to the AD pathology, and therapeutic restoration of ACh signaling is a common treatment strategy. How disease causation and the effect of ACh are linked to protein aggregation and neurotoxicity remains incompletely understood, thus limiting the development of more effective therapies. Here, we show that BAZ-2, the Caenorhabditis elegans ortholog of human BAZ2B, limits ACh signaling. baz-2 mutations reverse aggregation and toxicity of amyloid-beta as well as polyglutamine peptides, thereby restoring health and lifespan in nematode models of AD and HD, respectively. The neuroprotective effect of Δbaz-2 is mediated by choline acetyltransferase, phenocopied by ACh-esterase depletion, and dependent on ACh receptors. baz-2 reduction or ectopic ACh treatment augments proteostasis via induction of the endoplasmic reticulum unfolded protein response and the ubiquitin proteasome system.
Assuntos
Doença de Alzheimer , Doença de Huntington , Fatores Genéricos de Transcrição , Animais , Humanos , Acetilcolina/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas que Contêm Bromodomínio , Caenorhabditis elegans/metabolismo , Proteostase , Fatores Genéricos de Transcrição/metabolismoRESUMO
Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.
Assuntos
RNA , Fatores Genéricos de Transcrição , Iniciação da Transcrição Genética , RNA Polimerases Dirigidas por DNA/química , RNA/biossíntese , RNA Polimerase II/química , Fatores Genéricos de Transcrição/metabolismo , Humanos , Animais , Sus scrofa , Microscopia Crioeletrônica , Filmes CinematográficosRESUMO
The transcriptional machinery is thought to dissociate from DNA during replication. Certain proteins, termed epigenetic marks, must be transferred from parent to daughter DNA strands in order to maintain the memory of transcriptional states1,2. These proteins are believed to re-initiate rebuilding of chromatin structure, which ultimately recruits RNA polymerase II (Pol II) to the newly replicated daughter strands. It is believed that Pol II is recruited back to active genes only after chromatin is rebuilt3,4. However, there is little experimental evidence addressing the central questions of when and how Pol II is recruited back to the daughter strands and resumes transcription. Here we show that immediately after passage of the replication fork, Pol II in complex with other general transcription proteins and immature RNA re-associates with active genes on both leading and lagging strands of nascent DNA, and rapidly resumes transcription. This suggests that the transcriptionally active Pol II complex is retained in close proximity to DNA, with a Pol II-PCNA interaction potentially underlying this retention. These findings indicate that the Pol II machinery may not require epigenetic marks to be recruited to the newly synthesized DNA during the transition from DNA replication to resumption of transcription.
Assuntos
Cromatina , Replicação do DNA , DNA , Genes , RNA Polimerase II , Transcrição Gênica , Cromatina/genética , DNA/biossíntese , DNA/genética , DNA/metabolismo , DNA Polimerase II/metabolismo , Epigênese Genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Polimerase II/metabolismo , Fatores Genéricos de Transcrição/metabolismo , RNA/genética , RNA/metabolismoRESUMO
The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.
Assuntos
Optogenética , Fatores de Transcrição , Animais , Humanos , Camundongos , Regulação da Expressão Gênica , Células HEK293 , Mamíferos/genética , Mamíferos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Células CultivadasRESUMO
Plants perceive pathogens and induce robust transcriptional reprogramming to rapidly achieve immunity. The mechanisms of how immune-related genes are transcriptionally regulated remain largely unknown. Previously, the trihelix transcriptional factor ARABIDOPSIS SH4-RELATED 3 (ASR3) was shown to negatively regulate pattern-triggered immunity (PTI) in Arabidopsis thaliana. Here, we identified another trihelix family member ASR3-Interacting Transcriptional Factor 1 (AITF1) as an interacting protein of ASR3. ASR3-Interacting Transcriptional Factor 1 and ASR3 form heterogenous and homogenous dimers in planta. Both aitf1 and asr3 single mutants exhibited increased resistance against the bacterial pathogen Pseudomonas syringae, but the double mutant showed reduced resistance, suggesting AITF1 and ASR3 interdependently regulate immune gene expression and resistance. Overexpression of AITF1 triggered autoimmunity dependently on its DNA-binding ability and the presence of ASR3. Notably, autoimmunity caused by overexpression of AITF1 was dependent on a TIR-NBS-LRR (TNL) protein suppressor of AITF1-induced autoimmunity 1 (SAA1), as well as enhanced disease susceptibility 1 (EDS1), the central regulator of TNL signaling. ASR3-Interacting Transcriptional Factor 1 and ASR3 directly activated SAA1 expression through binding to the GT-boxes in SAA1 promoter. Collectively, our results revealed a mechanism of trihelix transcription factor complex in regulating immune gene expression, thereby modulating plant disease resistance and autoimmunity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Imunidade Vegetal , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Pseudomonas syringae/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismoRESUMO
Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.
Assuntos
Fatores Genéricos de Transcrição , Viroides , DNA/metabolismo , RNA/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Circular/genética , RNA Polimerase Dependente de RNA/genética , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Transcrição Gênica , Viroides/genética , Viroides/metabolismoRESUMO
BACKGROUND: General transcription factor IIi (GTF2I) mutations are very common in thymic epithelial tumors (TETs) and are related to a more favorable prognosis in TET patients. However, limited research has been conducted on the role of GTF2I in the tumor immune microenvironment (TIME). Further, long non-coding RNAs (lncRNAs) have been associated with the survival of patients with TETs. Therefore, this study aimed to explore the relationship between GTF2I mutations and TIME and build a new potential signature for predicting tumor recurrence in the TETs. Research data was downloaded from The Cancer Genome Atlas database and the CIBERSORT algorithm was used to evaluate TIME differences between GTF2I mutant and wild-type TETs. Relevant differentially expressed lncRNAs based on differentially expressed immune-related genes were identified to establish lncRNA pairs. We constructed a signature using univariate and multivariate Cox regression analyses. RESULTS: GTF2I is the most commonly mutated gene in TETs, and is associated with an increased number of early-stage pathological types, as well as no history of myasthenia gravis or radiotherapy treatment. In the GTF2I wild-type group, immune score and immune cell infiltrations with M2 macrophages, activated mast cells, neutrophils, plasma, T helper follicular cells, and activated memory CD4 T cells were higher than the GTF2I mutant group. A risk model was built using five lncRNA pairs, and the 1-, 3-, and 5-year area under the curves were 0.782, 0.873, and 0.895, respectively. A higher risk score was related to more advanced histologic type. CONCLUSION: We can define the GTF2I mutant-type TET as an immune stable type and the GTF2I wild-type as an immune stressed type. A signature based on lncRNA pairs was also constructed to effectively predict tumor recurrence.
Assuntos
Neoplasias Epiteliais e Glandulares , RNA Longo não Codificante , Fatores Genéricos de Transcrição , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Recidiva Local de Neoplasia/genética , Neoplasias Epiteliais e Glandulares/genética , Prognóstico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias do Timo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Microambiente TumoralRESUMO
Long noncoding RNAs (lncRNAs) have increasingly been recognized as being integral to cellular processes, including the antiviral immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is costly to the global swine industry. To identify PRRSV-related lncRNAs, we performed RNA deep sequencing and compared the profiles of lncRNAs in PRRSV-infected and uninfected Marc-145 cells. We identified a novel lncRNA called MAHAT (maintaining cell morphology-associated and highly conserved antiviral transcript; LTCON_00080558) that inhibits PRRSV replication. MAHAT binds and negatively regulates ZNF34 expression by recruiting and binding DDX6, an RNA helicase forming a complex with ZNF34. Inhibition of ZNF34 expression results in increased type I interferon expression and decreased PRRSV replication. This finding reveals a novel mechanism by which PRRSV evades the host antiviral innate immune response by downregulating the MAHAT-DDX6-ZNF34 pathway. MAHAT could be a host factor target for antiviral therapies against PRRSV infection. IMPORTANCE Long noncoding RNAs (lncRNAs) play important roles in viral infection by regulating the transcription and expression of host genes, and interferon signaling pathways. Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses in the swine industry worldwide, but the mechanisms of its pathogenesis and immunology are not fully understood. Here, a new lncRNA, designated MAHAT, was identified as a regulator of host innate immune responses. MAHAT negatively regulates the expression of its target gene, ZNF34, by recruiting and binding DDX6, an RNA helicase, forming a complex with ZNF34. Inhibition of ZNF34 expression increases type I interferon expression and decreases PRRSV replication. This finding suggests that MAHAT has potential as a new target for developing antiviral drugs against PRRSV infection.
Assuntos
Imunidade Inata , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , RNA Longo não Codificante , Replicação Viral , Animais , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Imunidade Inata/genética , Interferon Tipo I/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Suínos , Fatores Genéricos de Transcrição/metabolismo , Replicação Viral/genéticaRESUMO
Elongation factor TFIIS (transcription factor IIS) is structurally and biochemically probably the best characterized elongation cofactor of RNA polymerase II. However, little is known about TFIIS regulation or its roles during stress responses. Here, we show that, although TFIIS seems unnecessary under optimal conditions in Arabidopsis, its absence renders plants supersensitive to heat; tfIIs mutants die even when exposed to sublethal high temperature. TFIIS activity is required for thermal adaptation throughout the whole life cycle of plants, ensuring both survival and reproductive success. By employing a transcriptome analysis, we unravel that the absence of TFIIS makes transcriptional reprogramming sluggish, and affects expression and alternative splicing pattern of hundreds of heat-regulated transcripts. Transcriptome changes indirectly cause proteotoxic stress and deterioration of cellular pathways, including photosynthesis, which finally leads to lethality. Contrary to expectations of being constantly present to support transcription, we show that TFIIS is dynamically regulated. TFIIS accumulation during heat occurs in evolutionary distant species, including the unicellular alga Chlamydomonas reinhardtii, dicot Brassica napus and monocot Hordeum vulgare, suggesting that the vital role of TFIIS in stress adaptation of plants is conserved.
Assuntos
Arabidopsis , Fatores Genéricos de Transcrição , Arabidopsis/genética , Arabidopsis/fisiologia , Resposta ao Choque Térmico , RNA Polimerase II/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismoRESUMO
Tissue-specific basic helix-loop-helix (bHLH) transcription factors play an important role in cellular differentiation. We recently identified AmeloD as a tooth-specific bHLH transcription factor. However, the role of AmeloD in cellular differentiation has not been investigated. The aim of this study was to elucidate the role of AmeloD in dental epithelial cell differentiation. We found that AmeloD-knockout (AmeloD-KO) mice developed an abnormal structure and altered ion composition of enamel in molars, suggesting that AmeloD-KO mice developed enamel hypoplasia. In molars of AmeloD-KO mice, the transcription factor Sox21 encoding SRY-Box transcription factor 21 and ameloblast differentiation marker genes were significantly downregulated. Furthermore, overexpression of AmeloD in the dental epithelial cell line M3H1 upregulated Sox21 and ameloblast differentiation marker genes, indicating that AmeloD is critical for ameloblast differentiation. Our study demonstrated that AmeloD is an important transcription factor in amelogenesis for promoting ameloblast differentiation. This study provides new insights into the mechanisms of amelogenesis.
Assuntos
Ameloblastos , Dente , Fatores Genéricos de Transcrição/metabolismo , Ameloblastos/metabolismo , Amelogênese/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição/metabolismoRESUMO
Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.
Assuntos
Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Sumoilação , Fatores Genéricos de Transcrição/metabolismo , Transcrição Gênica , Cromatina/metabolismo , Humanos , Lisina/metabolismo , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores Genéricos de Transcrição/químicaRESUMO
The preinitiation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TATA-binding protein (TBP) and TBP-associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Iniciação da Transcrição Genética , Animais , Linhagem Celular , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Regulação Fúngica da Expressão Gênica , Humanos , Complexo Mediador/química , Complexo Mediador/genética , Camundongos , Regiões Promotoras Genéticas , Conformação Proteica , RNA Polimerase II/química , RNA Polimerase II/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores Genéricos de Transcrição/química , Fatores Genéricos de Transcrição/genética , Sítio de Iniciação de TranscriçãoRESUMO
Background: rs13405728 was identified as one of the most prevalent susceptibility loci for polycystic ovary syndrome (PCOS) in Han Chinese and Caucasian women. However, the target genes and potential mechanisms of the rs13405728 locus remain to be determined. Methods: Three-dimensional (3D) genome interactions from the ovary tissue were characterized via high-through chromosome conformation capture (Hi-C) and Capture Hi-C technologies to identify putative targets at the rs13405728 locus. Combined analyses of eQTL, RNA-Seq, DNase-Seq, ChIP-Seq, and sing-cell sequencing were performed to explore the molecular roles of these target genes in PCOS. PCOS-like mice were applied to verify the expression patterns. Results: Generally, STON1 and FSHR were identified as potential targets of the rs13405728 locus in 3D genomic interactions with epigenomic regulatory peaks, with STON1 (P=0.0423) and FSHR (P=0.0013) being highly expressed in PCOS patients. STON1 co-expressed genes were associated with metabolic processes (P=0.0008) in adipocytes (P=0.0001), which was validated in the fat tissue (P<0.0001) and ovary (P=0.0035) from fat-diet mice. The immune system process (GO:0002376) was enriched in FSHR co-expressed genes (P=0.0002) and PCOS patients (P=0.0002), with CD4 high expression in PCOS patients (P=0.0316) and PCOS-like models (P=0.0079). Meanwhile, FSHR expression was positively correlated with CD4 expression in PCOS patients (P=0.0252) and PCOS-like models (P=0.0178). Furthermore, androgen receptor (AR) was identified as the common transcription factor for STON1 and FSHR and positively correlated with the expression of STON1 (P=0.039) and FSHR (P=4e-06) in ovary tissues and PCOS-like mice. Conclusion: Overall, we identified STON1 and FSHR as potential targets for the rs13405728 locus and their roles in the processes of adipocyte metabolism and CD4 immune expression in PCOS, which provides 3D genomic insight into the pathogenesis of PCOS.
Assuntos
Proteínas de Membrana/genética , Síndrome do Ovário Policístico/genética , Receptores do FSH/genética , Fatores Genéricos de Transcrição/genética , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Antígenos CD4/imunologia , Feminino , Expressão Gênica , Loci Gênicos , Genoma , Humanos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Ovário/metabolismo , Síndrome do Ovário Policístico/imunologia , Síndrome do Ovário Policístico/metabolismo , Receptores Androgênicos/genética , Receptores do FSH/imunologia , Fatores Genéricos de Transcrição/metabolismoRESUMO
The Negative on TATA-less (NOT) 2_3_5 domain proteins play key roles in mRNA metabolism and transcription regulation, but few comprehensive studies have focused on this protein family in plants. In our study, a total of 30 NOT2_3_5 genes were identified in four cotton genomes: Gossypium. arboretum, G. raimondii, G. hirsutum and G. barbadense. Phylogenetic analysis showed that all the NOT2_3_5 domain proteins were divided into two classes. The NOT2_3_5 genes were expanded frequently, and segmental duplication had significant effects in their expansion process. The cis-regulatory elements analysis of NOT2_3_5 promoter regions indicated that NOT2_3_5 domain proteins might participate in plant growth and development processes and responds to exogenous stimuli. Expression patterns demonstrated that all of the GhNOT2_3_5 genes were expressed in the majority of tissues and fiber development stages, and that these genes were induced by multiple stresses. Quantitative real-time PCR showed that GbNOT2_3_5 genes were up-regulated in response to verticillium wilt and the silencing of GbNOT2_3_5-3/8 and GbNOT2_3_5-4/9 led to more susceptibility to verticillium wilt than controls. Identification and analysis of the NOT2_3_5 protein family will be beneficial for further research on their biological functions.
Assuntos
Resistência à Doença/genética , Gossypium , Proteínas de Plantas , Fatores Genéricos de Transcrição , Verticillium/crescimento & desenvolvimento , Estudo de Associação Genômica Ampla , Gossypium/genética , Gossypium/metabolismo , Gossypium/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismoRESUMO
The circadian clock matches various biological processes to diurnal environmental cycles, such as light and temperature. Accumulating evidence shows that chromatin modification is crucial for robust circadian oscillation in plants, although chromatin modifiers involved in regulating core clock gene expression have been limitedly investigated. Here, we report that the Jumonji C domain-containing histone demethylase JMJ29, which belongs to the JHDM2/KDM3 group, shapes rhythmic changes in H3K4me3 histone marks at core clock loci in Arabidopsis. The evening-expressed JMJ29 protein interacts with the Evening Complex (EC) component EARLY FLOWERING 3 (ELF3). The EC recruits JMJ29 to the CCA1 and PRR9 promoters to catalyze the H3K4me3 demethylation at the cognate loci, maintaining a low-level expression during the evening time. Together, our findings demonstrate that interaction of circadian components with chromatin-related proteins underlies diurnal fluctuation of chromatin structures to maintain circadian waveforms in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos , Desmetilação , Histonas/química , Fatores Genéricos de Transcrição/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatina , Regulação da Expressão Gênica de Plantas , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Fatores Genéricos de Transcrição/genéticaRESUMO
The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-ß1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-ß1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-ß1 stimulation. These results suggested that TGF-ß1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.
Assuntos
Ameloblastos/metabolismo , Germe de Dente/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Transcrição Gênica , Ameloblastos/efeitos dos fármacos , Animais , Diferenciação Celular , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Knockout , Morfogênese , Fosforilação , Ratos , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Germe de Dente/citologia , Germe de Dente/efeitos dos fármacos , Fatores Genéricos de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at a resolution below 4 angstroms. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.
Assuntos
Complexo Mediador/química , RNA Polimerase II/química , Fatores Genéricos de Transcrição/química , Iniciação da Transcrição Genética , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Humanos , Complexo Mediador/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.