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2.
Blood ; 138(18): 1648-1649, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34735000
4.
Nat Struct Mol Biol ; 28(10): 811-824, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34608337

RESUMO

The Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The inter-relatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics, we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.


Assuntos
Histonas/genética , Complexo Repressor Polycomb 1/genética , Iniciação da Transcrição Genética , Animais , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Histonas/metabolismo , Lisina/genética , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA Polimerase II/metabolismo , Análise de Célula Única
5.
Nat Commun ; 12(1): 6078, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34667177

RESUMO

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.


Assuntos
Neurônios/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos Knockout , Neurônios/química , Fosforilação , Domínios Proteicos , RNA/química , RNA/genética , RNA/metabolismo , RNA Polimerase II/genética , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Fatores de Transcrição/genética , Transcrição Genética
6.
Nat Commun ; 12(1): 5864, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620876

RESUMO

Pausing of RNA polymerase II (Pol II) close to promoters is a common regulatory step in RNA synthesis, and is coordinated by a ribonucleoprotein complex scaffolded by the noncoding RNA RN7SK. The function of RN7SK-regulated gene transcription in adult tissue homoeostasis is currently unknown. Here, we deplete RN7SK during mouse and human epidermal stem cell differentiation. Unexpectedly, loss of this small nuclear RNA specifically reduces transcription of numerous cell cycle regulators leading to cell cycle exit and differentiation. Mechanistically, we show that RN7SK is required for efficient transcription of highly expressed gene pairs with bidirectional promoters, which in the epidermis co-regulated cell cycle and chromosome organization. The reduction in transcription involves impaired splicing and RNA decay, but occurs in the absence of chromatin remodelling at promoters and putative enhancers. Thus, RN7SK is directly required for efficient Pol II transcription of highly transcribed bidirectional gene pairs, and thereby exerts tissue-specific functions, such as maintaining a cycling cell population in the epidermis.


Assuntos
Regulação da Expressão Gênica , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Pele/metabolismo , Transcrição Genética , Animais , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Cromatina , Montagem e Desmontagem da Cromatina , Epiderme , Feminino , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Splicing de RNA , Pele/patologia , Células-Tronco
8.
Mol Cell ; 81(17): 3443-3445, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34478651

RESUMO

Complementary papers by Nguyen et al. (2021) and Baek et al. (2021) track the assembly of the pre-initiation complexes at gene promoters using single-molecule microscopy, revealing dynamic spatiotemporal regulation of transcription initiation.


Assuntos
RNA Polimerase II , Imagem Individual de Molécula , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo
9.
Elife ; 102021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34515029

RESUMO

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 Transcrição
10.
Chem Commun (Camb) ; 57(75): 9558-9561, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477193

RESUMO

Amanitin is used extensively as a research tool to inhibit RNA Pol II thereby implicating its role in mRNA transcription. Recently, amanitin has gained traction as a toxic payload for targeted therapy. Here we report the first-ever photocaged amanitin analog, that is non-toxic and can be pre-loaded into cells. Light provides a means to inhibit RNA Pol II and provoke cell death on-demand.


Assuntos
Amanitinas/farmacologia , Pró-Fármacos/farmacologia , RNA Polimerase II/antagonistas & inibidores , Amanitinas/síntese química , Amanitinas/química , Animais , Células CHO , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Relação Dose-Resposta a Droga , Estrutura Molecular , Processos Fotoquímicos , Pró-Fármacos/síntese química , Pró-Fármacos/química , RNA Polimerase II/metabolismo
11.
Nature ; 598(7880): 368-372, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34526721

RESUMO

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.


Assuntos
Microscopia Crioeletrônica , Reparo do DNA , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Transcrição Genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação
12.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544872

RESUMO

The bZIP transcription factor ATF6α is a master regulator of endoplasmic reticulum (ER) stress response genes. In this report, we identify the multifunctional RNA polymerase II transcription factor Elongin as a cofactor for ATF6α-dependent transcription activation. Biochemical studies reveal that Elongin functions at least in part by facilitating ATF6α-dependent loading of Mediator at the promoters and enhancers of ER stress response genes. Depletion of Elongin from cells leads to impaired transcription of ER stress response genes and to defects in the recruitment of Mediator and its CDK8 kinase subunit. Taken together, these findings bring to light a role for Elongin as a loading factor for Mediator during the ER stress response.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Elonguina/metabolismo , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Fator 6 Ativador da Transcrição/genética , Animais , Elonguina/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Células HeLa , Humanos , Complexo Mediador/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Ratos , Transdução de Sinais , Ativação Transcricional
13.
J Biol Chem ; 297(4): 101205, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34543624

RESUMO

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.


Assuntos
Chaperonas de Histonas/metabolismo , Metiltransferases/metabolismo , Elementos de Resposta , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Cromatina/genética , Cromatina/metabolismo , Chaperonas de Histonas/genética , Espectrometria de Massas , Metiltransferases/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Elongação da Transcrição/genética
14.
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
15.
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
16.
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
17.
DNA Repair (Amst) ; 106: 103192, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34358806

RESUMO

Transcription-blocking DNA lesions (TBLs) in genomic DNA are triggered by a wide variety of DNA-damaging agents. Such lesions cause stalling of elongating RNA polymerase II (RNA Pol II) enzymes and fully block transcription when unresolved. The toxic impact of DNA damage on transcription progression is commonly referred to as transcription stress. In response to RNA Pol II stalling, cells activate and employ transcription-coupled repair (TCR) machineries to repair cytotoxic TBLs and resume transcription. Increasing evidence indicates that the modification and processing of stalled RNA Pol II is an integral component of the cellular response to and the repair of TBLs. If TCR pathways fail, the prolonged stalling of RNA Pol II will impede global replication and transcription as well as block the access of other DNA repair pathways that may act upon the TBL. Consequently, such prolonged stalling will trigger profound genome instability and devastating clinical features. In this review, we will discuss the mechanisms by which various types of TBLs are repaired by distinct TCR pathways and how RNA Pol II processing is regulated during these processes. We will also discuss the clinical consequences of transcription stress and genotype-phenotype correlations of related TCR-deficiency disorders.


Assuntos
Dano ao DNA , Reparo do DNA , Instabilidade Genômica , RNA Polimerase II/metabolismo , Transcrição Genética , Envelhecimento , Animais , DNA/metabolismo , Eucariotos/enzimologia , Eucariotos/genética , Eucariotos/metabolismo , Humanos
18.
Mol Cell ; 81(17): 3576-3588.e6, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34384542

RESUMO

RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.


Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética/fisiologia , Núcleo Celular/metabolismo , Complexo Mediador/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , TATA Box/genética , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Transcrição Genética/genética
19.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445207

RESUMO

Recent studies show a crucial role of post-transcriptional processes in the regulation of gene expression. Our research has shown that mRNA retention in the nucleus plays a significant role in such regulation. We studied larch microsporocytes during meiotic prophase, characterized by pulsatile transcriptional activity. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm but are retained in the cell nucleus and especially in Cajal bodies, where non-fully-spliced transcripts with retained introns are accumulated. Analysis of the transcriptome of these cells and detailed analysis of the nuclear retention and transport dynamics of several mRNAs revealed two main patterns of nuclear accumulation and transport. The majority of studied transcripts followed the first one, consisting of a more extended retention period and slow release to the cytoplasm. We have shown this in detail for the pre-mRNA and mRNA encoding RNA pol II subunit 10. In this pre-mRNA, a second (retained) intron is posttranscriptionally spliced at a precisely defined time. Fully mature mRNA is then released into the cytoplasm, where the RNA pol II complexes are produced. These proteins are necessary for transcription in the next pulse to occur.mRNAs encoding translation factors and SERRATE followed the second pattern, in which the retention period was shorter and transcripts were rapidly transferred to the cytoplasm. The presence of such a mechanism in various cell types from a diverse range of organisms suggests that it is an evolutionarily conserved mechanism of gene regulation.


Assuntos
Núcleo Celular/metabolismo , Larix/metabolismo , Pólen/metabolismo , Prófase , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Núcleo Celular/genética , Larix/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética
20.
PLoS Comput Biol ; 17(8): e1009256, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34383743

RESUMO

Metazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters.


Assuntos
Genoma Humano , Regiões Promotoras Genéticas , Algoritmos , Animais , Sequência de Bases , Biologia Computacional , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Modelos Genéticos , Modelos Estatísticos , RNA Polimerase II/metabolismo , Especificidade da Espécie , TATA Box , Transcrição Genética
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