RESUMO
TAF4b is a cell type-specific subunit of the general transcription factor TFIID. Here, we show that TAF4b is highly expressed in embryonic stem cells (ESC) and is down-regulated upon differentiation. To examine the role of TAF4b in ESC, we applied a knockdown (KD) approach. TAF4b depletion is associated with morphological changes and reduced expression of the self-renewal marker alkaline phosphatase. In contrast, KD of TAF4, a ubiquitously expressed TAF4b paralog, retained and even stabilized ESC stemness. Retinoic acid-induced differentiation was facilitated in the absence of TAF4b but was significantly delayed by TAF4 KD. Furthermore, TAF4b supports, whereas TAF4 inhibits, ESC proliferation and cell cycle progression. We identified a subset of TAF4b target genes preferentially expressed in ESC and controlling the cell cycle. Among them are the germ cell-specific transcription factor Sohlh2 and the protein kinase Yes1, which was recently shown to regulate ESC self-renewal. Interestingly, Sohlh2 and Yes1 are also targets of the pluripotency factor Oct4, and their regulation by Oct4 is TAF4b-dependent. Consistent with that, TAF4b but not TAF4 interacts with Oct4. Our findings suggest that TAF4b cooperates with Oct4 to regulate a subset of genes in ESC, whereas TAF4 is required for later embryonic developmental stages.
Assuntos
Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Camundongos , Mutação , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas Proto-Oncogênicas c-yes/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Transcrição GênicaRESUMO
The proximal promoter consists of binding sites for transcription regulators and a core promoter. We identified an overrepresented motif in the proximal promoter of human genes with an Initiator (INR) positional bias. The core of the motif fits the INR consensus but its sequence is more strict and flanked by additional conserved sequences. This strict INR (sINR) is enriched in TATA-less genes that belong to specific functional categories. Analysis of the sINR-containing DHX9 and ATP5F1 genes showed that the entire sINR sequence, including the strict core and the conserved flanking sequences, is important for transcription. A conventional INR sequence could not substitute for DHX9 sINR whereas, sINR could replace a conventional INR. The minimal region required to create the major TSS of the DHX9 promoter includes the sINR and an upstream Sp1 site. In a heterologous context, sINR substituted for the TATA box when positioned downstream to several Sp1 sites. Consistent with that the majority of sINR promoters contain at least one Sp1 site. Thus, sINR is a TATA-less-specific INR that functions in cooperation with Sp1. These findings support the idea that the INR is a family of related core promoter motifs.
Assuntos
Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Cricetinae , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Fator de Transcrição Sp1/metabolismo , TATA Box , Fator de Transcrição TFIID/fisiologia , Sítio de Iniciação de Transcrição , Ativação Transcricional , Fator de Transcrição YY1/metabolismoRESUMO
The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b.TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is approximately 70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes.
Assuntos
DNA/metabolismo , Regiões Promotoras Genéticas/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Bovinos , Linhagem Celular , Imunoprecipitação da Cromatina , DNA/genética , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , TransfecçãoRESUMO
A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNA-contacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.