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.

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.

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, TFs from the same DBD family can display distinct binding behaviors during mitosis. 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 globally decreased. Surprisingly, live-cell imaging shows that both factors appear excluded from mitotic chromosomes, 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 HSF2 DBD alone can coat mitotic chromosomes, but is insufficient to promote HSF1 coating. 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.

No abstract

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). Decades of research have shown that TBP is essential for proper transcription by the three RNA Pols, though the emergence of TBP paralogs throughout evolution have expanded the complexity in RNA Pol initiation. We have previously shown that acute TBP depletion in mouse embryonic stem cells led to a global decrease in Pol III activity, consistent with the requirement of TBP in Pol III initiation. In contrast, Pol II transcription remained unaffected in the absence of TBP and its paralogs. In this report, we show that, in contrast to Pol II-transcribed genes, the TBP paralog TRF2 does not bind to Pol I promoters, and therefore cannot functionally replace TBP upon depletion. Importantly, acute TBP depletion has no major effect on 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.