RESUMO
The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.
Assuntos
Mitose , Regiões Promotoras Genéticas , RNA Polimerase I , Proteína de Ligação a TATA-Box , Transcrição Gênica , Animais , RNA Polimerase I/metabolismo , RNA Polimerase I/genética , Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Ligação Proteica , DNA Ribossômico/genética , DNA Ribossômico/metabolismoRESUMO
Transcription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the pre-initiation complex onto promoter DNA. Decades of research have shown that the TATA-box binding protein (TBP) is essential for Pol II loading and initiation. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells has no global effect on ongoing Pol II transcription. In contrast, acute TBP depletion severely impairs RNA Polymerase III initiation. Furthermore, Pol II transcriptional induction occurs normally upon TBP depletion. This TBP-independent transcription mechanism is not due to a functional redundancy with the TBP paralog TRF2, though TRF2 also binds to promoters of transcribed genes. Rather, we show that the TFIID complex can form and, despite having reduced TAF4 and TFIIA binding when TBP is depleted, the Pol II machinery is sufficiently robust in sustaining TBP-independent transcription.
Assuntos
RNA Polimerase II , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , TATA Box/genética , Células-Tronco Embrionárias/metabolismo , Transcrição Gênica , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , RNA Polimerase III/genéticaRESUMO
For over two decades, scientists have observed that most transcription factors (TFs) become excluded from mitotic chromosomes of mammalian cells undergoing cell division. The few TFs that were observed to remain bound to chromosomes have been termed mitotic bookmarkers and were predicted to play important roles in reestablishing transcription after mitosis. Using live-cell imaging of endogenous TFs in mouse embryonic stem cells, we discovered that the observed exclusion from mitotic chromosomes is largely a result of formaldehyde cross-linking and that in fact, most TFs bind to mitotic chromosomes throughout mitosis. Here, we describe the single-molecule live-cell imaging and analytical tools we used to characterize and quantify TF diffusion and binding as mouse embryonic stem cells proceed through mitosis.