RNA Polymerase II transcription independent of TBP in murine embryonic stem cells

  1. James ZJ Kwan
  2. Thomas F Nguyen
  3. Anuli C Uzozie
  4. Marek A Budzynski
  5. Jieying Cui
  6. Joseph MC Lee
  7. Filip Van Petegem
  8. Philipp F Lange
  9. Sheila S Teves

  • Curated by eLife

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    eLife assessment

    This important study employs auxin-induced degradation to show that the TATA-binding protein (TBP) is not required for ongoing RNA polymerase II transcription nor heat-shock or retinoic acid-induced transcription, but that TBP is essential for RNA polymerase III transcription, with TBP-independent TFIID complexes being assembled and present at the transcription start sites of polymerase II-transcribed promoters. The evidence for the major claims is currently incomplete.

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Abstract

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.

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  1. Version published to 10.7554/elife.83810 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This study addresses the role of the general transcription factor TBP (TATA-binding protein), a subunit of the TFIID complex, in RNA polymerase II transcription. While TBP has been described as a key component of protein complexes involved in transcription by all three RNA polymerases, several previous studies on TBP loss of function and on the function of its TRF2 and TRF3 paralogues have questioned its essential role in RNA polymerase II transcription. This new study uses auxin induced TBP degradation in mouse ES cells to provide strong evidence that its loss does not affect ongoing polymerase II transcription or heat-shock and retinoic acid-induced transcription activation, but severely inhibits polymerase III transcription. The authors coupled TBP degradation with TRF2 knock out to show that it does not account for the residual TBP-independent transcription. Rather the study provides evidence that TFIID can assemble and is recruited to promoters in the absence of TBP.

    All together the study provides compelling evidence for TBP-independent polymerase II transcription, but a better characterization of the residual TFIID complex and recruitment of other general transcription factors to promoters would strengthen the conclusions.

    We thank the reviewer for their accurate summary of our findings and the public assessment of our manuscript.

    Reviewer #2 (Public Review):

    The paper is intriguing, but to me, a main weakness is that the imaging experiments are done with overexpressed protein. Another is that the different results for the different subunits of TFIID would indicate that there are multiple forms of TFIID in the nucleus, which no one has observed/proposed before. Otherwise, the experimental data would have to be interpreted in a more nuance way. Additionally, there is no real model of how a TBP-depleted TFIID would recruit Pol II. Do the authors suggest that when TBP is present, it is not playing a role in Pol II transcription, despite being at all promoters? Or that in its absence an alternative mechanism takes over? In the latter case, are they proposing that it is just based on the rest of TFIID? How? The authors do not provide a mechanistic explanation of what is actually happening and how Pol II is being recruited to promoters.

    We thank the reviewer for their public review of our manuscript. Although the reviewer poses many interesting questions raised from our findings, they would be a great focus for future directions.

    We agree that our imaging experiments using over-expressed constructs have limitations. Though they provide insight that is unique and orthogonal to the genomics analyses, we agree that they are still preliminary, and therefore we have removed them from the manuscript, with the hope of further developing these experiments into a follow-up manuscript.

    While we cannot exclude different forms of TFIID in the cell, previous studies have identified different TAF-containing complexes. Indeed, we referenced several of these studies in our manuscript, including TFTC/SAGA. Furthermore, in our Discussion section, we speculated how a large multi-subunit complex like TFIID may not behave as a monolith but rather have distinct dynamics/behavior among the subunits. Some studies are now revealing that biochemically defined complexes behave more as a hub, with subunits having distinct dynamics coming in and out of the complex, but in a way such that a snapshot at any given time would show a stably formed complex.

    What TBP does for Pol II is an intriguing question, and one that we had thought we could answer with our rapid depletion system. One possibility is that Pol II initiation has evolved to have so many redundant mechanisms such that removal of one factor (TBP) would not disrupt the whole system. And yet, TBP remains a highly essential gene (perhaps mostly for its essential role in Pol III transcription), and therefore, its binding to Pol II gene promoters has been maintained, almost in a vestigial way. Of course, this is speculative, and our rapid depletion system only shows us that TBP is not required for Pol II transcription, not what it does when it binds to promoters.

    Lastly, we believe that our study tested 3 potential mechanisms that could explain TBP-independence for Pol II transcription. 1) We tested the possibility that TBP is only needed for induction and not for subsequent re-initiation. We provide evidence using two orthogonal induction systems that this is not the case. 2) We tested whether the TRF2 paralog could functionally replace TBP, and show that this is also not the case. 3) We show that TFIID can form in the absence of TBP. While we agree that there are more mechanisms to test, addressing all of them would require a re-examination of over 50 years of research that would not be feasible to report in a single manuscript, especially for a system as complex as Pol II initiation.

    Reviewer #3 (Public Review):

    In this study, the authors set out to study the requirement of the TATA binding protein (TBP) in transcription initiation in mESCs. To this end they used an auxin inducible degradation (AID) system. They report that by using the AID-TBP system after auxin degradation, 10-20% of TBP protein is remaining in mESCs. The authors claim that as, the observed 80-90% decrease of TBP levels are not accompanied by global changes in RNA polymerase II (Pol II) chromatin occupancy or nascent mRNA levels, TBP is not required for Pol II transcription. In contrast, they find that under similar TBP-depletion conditions tRNA transcription and Pol III chromatin occupancy were impaired. The authors also asked whether the mouse TBP paralogue, TBPL1 (also called TRF2) could functionally replace TBP, but they find that it does not. From these and additional experiments the authors conclude that redundant mechanisms may exist in which TBP-independent TFIID like complexes may function in Pol II transcription.

    The major strengths of this manuscript are the numerous genome-wide investigations, such as many different CUT&Tag experiments, and NET-seq experiments under control and +auxin conditions and their analyses. Weaknesses lie in some experimental setups (i.e. overexpression of Halo-tagged TAFs), mainly in the overinterpretation (or misinterpretation) of the data and in the lack of a fair discussion of the obtained data in comparison to observations described in the literature. As a result, very often the interpretation of data does not fully support the conclusions. Nevertheless, the findings that 80-90% decrease in cellular TBP levels do not have a major effect on Pol II transcription are interesting, but the manuscript needs some tuning down of many of the authors' very strong conclusions, correcting several weaker points and with a more careful and eventually more interesting Discussion.

    We thank the reviewer for their public review of our manuscript. We would like to add that, in addition to testing the TBP paralog for redundancy, we also tested a mechanism in which TBP would be required for the initial round of transcription but not for subsequent ones. We show that data from orthogonal experiments that this mechanism is not the case. As in our response to Reviewer 2, we agree that our over-expression imaging experiments are still somewhat preliminary, and therefore we have removed these experiments and potential over/misinterpretation of these results from the manuscript.

  3. eLife

    eLife assessment

    This important study employs auxin-induced degradation to show that the TATA-binding protein (TBP) is not required for ongoing RNA polymerase II transcription nor heat-shock or retinoic acid-induced transcription, but that TBP is essential for RNA polymerase III transcription, with TBP-independent TFIID complexes being assembled and present at the transcription start sites of polymerase II-transcribed promoters. The evidence for the major claims is currently incomplete.

  4. eLife

    Reviewer #1 (Public Review):

    This study addresses the role of the general transcription factor TBP (TATA-binding protein), a subunit of the TFIID complex, in RNA polymerase II transcription. While TBP has been described as a key component of protein complexes involved in transcription by all three RNA polymerases, several previous studies on TBP loss of function and on the function of its TRF2 and TRF3 paralogues have questioned its essential role in RNA polymerase II transcription. This new study uses auxin induced TBP degradation in mouse ES cells to provide strong evidence that its loss does not affect ongoing polymerase II transcription or heat-shock and retinoic acid-induced transcription activation, but severely inhibits polymerase III transcription. The authors coupled TBP degradation with TRF2 knock out to show that it does not account for the residual TBP-independent transcription. Rather the study provides evidence that TFIID can assemble and is recruited to promoters in the absence of TBP.

    All together the study provides compelling evidence for TBP-independent polymerase II transcription, but a better characterization of the residual TFIID complex and recruitment of other general transcription factors to promoters would strengthen the conclusions.

  5. eLife

    Reviewer #2 (Public Review):

    The paper is intriguing, but to me, a main weakness is that the imaging experiments are done with overexpressed protein. Another is that the different results for the different subunits of TFIID would indicate that there are multiple forms of TFIID in the nucleus, which no one has observed/proposed before. Otherwise, the experimental data would have to be interpreted in a more nuance way. Additionally, there is no real model of how a TBP-depleted TFIID would recruit Pol II. Do the authors suggest that when TBP is present, it is not playing a role in Pol II transcription, despite being at all promoters? Or that in its absence an alternative mechanism takes over? In the latter case, are they proposing that it is just based on the rest of TFIID? How? The authors do not provide a mechanistic explanation of what is actually happening and how Pol II is being recruited to promoters.

  6. eLife

    Reviewer #3 (Public Review):

    In this study, the authors set out to study the requirement of the TATA binding protein (TBP) in transcription initiation in mESCs. To this end they used an auxin inducible degradation (AID) system. They report that by using the AID-TBP system after auxin degradation, 10-20% of TBP protein is remaining in mESCs. The authors claim that as, the observed 80-90% decrease of TBP levels are not accompanied by global changes in RNA polymerase II (Pol II) chromatin occupancy or nascent mRNA levels, TBP is not required for Pol II transcription. In contrast, they find that under similar TBP-depletion conditions tRNA transcription and Pol III chromatin occupancy were impaired. The authors also asked whether the mouse TBP paralogue, TBPL1 (also called TRF2) could functionally replace TBP, but they find that it does not. From these and additional experiments the authors conclude that redundant mechanisms may exist in which TBP-independent TFIID like complexes may function in Pol II transcription.

    The major strengths of this manuscript are the numerous genome-wide investigations, such as many different CUT&Tag experiments, and NET-seq experiments under control and +auxin conditions and their analyses. Weaknesses lie in some experimental setups (i.e. overexpression of Halo-tagged TAFs), mainly in the overinterpretation (or misinterpretation) of the data and in the lack of a fair discussion of the obtained data in comparison to observations described in the literature. As a result, very often the interpretation of data does not fully support the conclusions.
    Nevertheless, the findings that 80-90% decrease in cellular TBP levels do not have a major effect on Pol II transcription are interesting, but the manuscript needs some tuning down of many of the authors' very strong conclusions, correcting several weaker points and with a more careful and eventually more interesting Discussion.

  7. Version published to 10.1101/2021.03.28.437425v1 on bioRxiv