RESUMEN
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.
Asunto(s)
Mitosis , Regiones Promotoras Genéticas , ARN Polimerasa I , Proteína de Unión a TATA-Box , Transcripción Genética , Animales , ARN Polimerasa I/metabolismo , ARN Polimerasa I/genética , Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Unión Proteica , ADN Ribosómico/genética , ADN Ribosómico/metabolismoRESUMEN
A large number of transcription factors have been shown to bind and interact with mitotic chromosomes, which may promote the efficient reactivation of transcriptional programs following cell division. Although the DNA-binding domain (DBD) contributes strongly to TF behavior, the mitotic behaviors of TFs from the same DBD family may vary. To define the mechanisms governing TF behavior during mitosis in mouse embryonic stem cells, we examined two related TFs: Heat Shock Factor 1 and 2 (HSF1 and HSF2). We found that HSF2 maintains site-specific binding genome-wide during mitosis, whereas HSF1 binding is somewhat decreased. Surprisingly, live-cell imaging shows that both factors appear excluded from mitotic chromosomes to the same degree, and are similarly more dynamic in mitosis than in interphase. Exclusion from mitotic DNA is not due to extrinsic factors like nuclear import and export mechanisms. Rather, we found that the HSF DBDs can coat mitotic chromosomes, and that HSF2 DBD is able to establish site-specific binding. These data further confirm that site-specific binding and chromosome coating are independent properties, and that for some TFs, mitotic behavior is largely determined by the non-DBD regions.
Asunto(s)
Cromosomas , Proteínas de Choque Térmico , Mitosis , Factores de Transcripción , Animales , Ratones , Cromosomas/genética , Cromosomas/metabolismo , ADN/metabolismo , Factores de Transcripción del Choque Térmico/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
ARN Polimerasa II , Factores de Transcripción , Animales , Ratones , Factores de Transcripción/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , TATA Box/genética , Células Madre Embrionarias/metabolismo , Transcripción Genética , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , ARN Polimerasa III/genéticaRESUMEN
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.
