Non-canonical TAF complexes regulate active promoters in human embryonic stem cells

1. The reviewer commented that, if the information were available, the results of RNA polymerase II and TAF recruitment would be a useful addition to the shRNA-mediated knockdown experiments of Figure 5.

For different reasons, as explained below, neither of the two datasets mentioned by the reviewer is available.

With regard to RNA polymerase II recruitment, a number of previous studies by our laboratory and others have analyzed the relationship between TAF dependence and RNA polymerase II occupancy in vivo. Notably, these studies have been carried out in a wide range of eukaryotes including yeast, Drosophila S2 cells and mammalian cells. The results of these studies have clearly demonstrated that when a TAF is required for transcription of a gene, loss-of-function of that TAF (resulting from mutational inactivation, RNA interference-mediated depletion or decreased expression) results in diminished transcription that is accompanied by decreased RNA polymerase II occupancy in a chromatin-immunoprecipitation (ChIP) assay. Representative examples of such studies include those performed in yeast (Shen et al. 2003, EMBO J. 22:3395; Sharma et al. 2003, Genes Dev. 17:502), Drosophila S2 cells (Vorobyeva et al. 2009, PNAS 106:11049) and mammalian cells (Deato and Tjian 2007, Genes Dev. 21:2137; Zaborowska et al. 2012, Transcription 3:92). In addition to these in vivo examples, several in vitro studies have also demonstrated a role for TAFs in RNA polymerase II recruitment (see, for examples, Wu and Chiang 2001, J. Biol. Chem. 276:34235; Felinski and Quinn 2001, PNAS 98:13078).

Therefore, because the relationship between TAF dependence and RNA polymerase II occupancy had been well established, this issue was not investigated in our current study.

With regard to TAF recruitment, we are in fact actively studying the requirement of specific TAFs for recruitment of other TAFs and TBP at class I and class II promoters. We believe that when these results are completed they may provide insights into the roles of specific TAFs in transcription of class I and class II genes and shed light on the differences between class I and class II promoters. However, our preliminary results indicate that the requirement of specific TAFs for recruitment of other TAFs and TBP is complex, differs among the TAFs, and may also differ between promoters of the same class. Therefore, until we have performed a comprehensive analysis, which will require considerably more experimentation and time and may necessitate genome-wide studies, we think it would premature to publish a limited set of results, which could be potentially non-representative and misleading.

2. The reviewer requested that we revise Figure 6E so that the Y-axis ranges from 0 to 100 percent to provide a better perspective on the data.

We have revised the figure as requested by the reviewer.

3. The reviewer requested that we revise Figure 6F so that the Y-axis ranges from 0.0 to 1.0. He/she also asked that we explain the criteria used to determine whether or not a gene has an alternative promoter.

We have revised the figure as requested by the reviewer. The criteria we used to determine whether a gene has an alternative promoter is described in detail in the Materials and Methods section. Prompted by the reviewer's comment, we have revised the main text describing Figure 6F to briefly state that alternative promoters were identified based on UCSC Genome browser annotations, and refer the reader to the Materials and Methods section for additional details.