Peer review of: Taf14 recognizes a common motif in transcriptional machineries and facilitates their clustering by (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript, Chen and coworkers provide a structural and functional characterization of the 
Taf14 extra-terminal (ET) domain in isolation and in complex with various coactivator proteins. 
From the structural and functional studies, the authors conclude that disorder and the potential for 
multivalent interactions of Taf14 enables Taf14 to function as a molecular hub to coordinate 
transcription. Furthermore, the authors report that Taf14 is able to undergo liquid-liquid phase 
separation alone and in complex with its binding partners. This type of behavior has been observed 
in a multitude of other systems involving disordered proteins and transcriptional regulators and is 
not quite surprising, especially with the use of crowding agents as employed here. However, the 
observation that Taf14 ET undergoes a co-folding process upon binding to other disordered peptide 
sequences containing a common motif is very interesting and expands our current views of how 
transcriptional regulation is achieved via hub proteins. The data are presented clearly and 
convincingly, and this manuscript should be of broad interest to the readers of Nature 
Communications. I suggest some additional experiments and minor comments below that should 
improve the quality of the manuscript, address some technical concerns, and bolster the authors’ 
claims of intrinsic disorder in the Taf14 ET domain. 

1. There is a potential alternate explanation for the appearance of the NMR spectrum of the TAF14 
ET domain in isolation (Fig. 1D). It appears that far fewer resonances are observed than would be 
expected based on the sequence of the construct used for this experiment. This spectrum could 
also represent a soluble aggregate, where the only resonances that are observable are those that 
remain dynamic and are not subject to size-induced broadening. What concentration of 15N-
labeled Taf14 ET was used to obtain the spectrum shown? To probe potential self-association of 
Taf14 ET (which would be consistent with the microscopy data), the authors could collect NMR 
spectra at a range of concentrations, to observe whether the spectra improve (more peaks, better 
resolution) at lower concentrations. Additionally, CD spectra of Taf14 ET should be obtained for 
Taf14 ET in isolation and in complex with its binding partners to determine whether Taf14 ET is 
indeed disordered in its unbound state, or whether it is sampling a more molten globular/semi-
structured state in solution. 

2. A point of confusion—different Kd values are given for Sth1 in Fig. 1C and Fig. 4A. Were these 
values obtained for the same construct of Sth1? For purposes of comparison with the other binding 
partners, for which minimal constructs were used, it would be helpful to have a comparison to a 
peptide containing the minimal binding motif of Sth1 (ie. the ordered segment identified from the 
NMR structure). It is important to make consistent comparisons between the various binding 
partners, and despite the disordered nature of the flanking regions in the Sth1 construct, these 
regions have been shown in many recent papers to have an influence on binding affinity complex 
stability. 

Other general comments: 
- Error bars are largely missing in the presentation of the experimental data. For ITC experiments, 
how are the uncertainties in Kd values estimated? Why are there no uncertainties specified for the 
thermodynamic parameters? 
- Thermodynamic parameters should be represented in kJ/mol. 
- Pay attention to significant figures. 

Reviewer #2 (Remarks to the Author): 

Review of “The extra-terminal domain of Taf14 recognizes a common motif in diverse 
transcriptional machineries and facilitates their clustering by phase separation” 

In this manuscript, Chen et al. interrogate the mechanism by which the YEATS containing, 
chromatin binding protein Taf14 coordinates interactions between itself and the up to 7 distinct 
complexes with which it interacts in the S. cerevisiae model organism. Taf14, along with its 
metazoan ortholog Af9, interact with acylated histone proteins to facilitate a gene regulatory 
response. Through structural biology approaches and detailed biophysical measurements, the 
authors identify a common peptide motif present in subunits of each of six distinct transcription-
related complexes to which Taf14 binds. As a test case, the authors make a separation of function 
mutant in Sth1, a component of the RSC chromatin remodeling complex, to reduce binding 
between Sth1 and Taf14 without disrupting the RSC complex. This mutant displays both growth 
defects as well as alterations in gene regulation. Finally, the authors demonstrate that Taf14, 
under protein crowding conditions, can form phase separated liquid droplets. Furthermore, these 
droplets can partition peptides from distinct transcription complexes into the same phase 
separated liquid droplet. This observation led the authors to propose a mechanism for Taf14 
function: through its YEATS domain acylated histone binding activity, Taf14 partitions to specific 
genomic locations to regulate transcription; and through phase separation and its ability to bind 
multiple transcription-related complexes, Taf14 coordinates the activities of these 7 different 
transcription-related complexes in a phase separated and concentrated location, similar to 
mechanisms observed in metazoan super-enhancers. The data presented in the submitted 
manuscript are compelling and the interpretations described by the authors are consistent with the 
data. In addition, the study is of interest to the broader transcription regulation field and timely in 
its findings. Therefore, with minor revisions addressed below, it is the opinion of this reviewer that 
the manuscript be accepted for publication: 

1. Either the methods section or the figure legends should include more information regarding 
experimental reproducibility. The number of times each experiment was performed should be 
included in these descriptions. Specifically, the number of biological replicates used for growth 
assays and the number of times the coIP experiments were performed. 

2. Either commentary or additional experiments should be provided to demonstrate that 
interactions between Taf14 and the intact protein complexes are abrogated upon mutation of the 
extra terminal binding domains instead of just relying on peptide interaction assays. In the ITC 
experiments, binding between the Sth1 peptide and Taf14 is completely abrogated in the m4 
mutant. However, the coIP experiment only shows a reduction of Taf14 binding, not an 
elimination. While the coIP data is supported by growth data, qPCR and RNAseq analyses, it could 
be bolstered with quantitation of the coIP analyses including multiple replicates. Furthermore, the 
authors specifically mention that Tfg1 contains two distinct sequences that can interact with Taf14. 
Taf2 was shown to have multiple domains that can interact with Taf14 in a cited paper. In the full 
length Taf2 in the context of TFIID, mutation of either of the domains was not sufficient to 
abrogate binding between Taf14 and TFIID. While the identified peptide sequence in the EBD 
obviously contributes to Taf14 binding in the peptides tested, without testing of the intact protein, 
it is not possible to show that this peptide is essential for interaction for each of the different 
complexes. Considering this type of analysis was performed for Sth1 and RSC, the absence of 
these experiments should not preclude publication. 

3. In line 205, when describing the temperature of the heat sensitivity, the authors claim that the 
cells grew normally below 32oC. However, this temperature was not tested, as shown in figure 3. 
This data point should be included in the figure or the text should be amended to “below 30oC”. In 
addition, the sth1m4 mutant appears to have a modestly slower growth phenotype compared to 
the WT even at lower temperatures. Growth curves, while not necessary for this study, could 
provide a more quantitative reading of growth rates comparing the two strains. 

4. The phase separated liquid droplet data would be even more compelling if the mutant peptides 
were also included in the analyses. This would demonstrate that the partitioning of the peptides to 
the Taf14 droplets is dependent on the amino acids required for nanomolar binding affinity. 


Reviewer #3 (Remarks to the Author): 

Chen et al. report that the ET domain of TAF-14 binds a common motif (EBM) in unstructured 
sequences of several nuclear factors, as judged by GST pulldown assays. They solve the solution 
structure of the Taf14 ET domain with the EBM of Sth1. Based on the structure, they design 
mutants disrupting this interaction and analyse their affinity by ITC. They further find similar 
interaction patterns for several other EBM peptides by ITC. 
Next, they use a quadruple Sth1 mutant that completely abolishes interaction with Taf14 in ITC. 
They find that in yeast cells, if this mutant is the only copy expressed, it strongly diminishes 
interaction with Taf14, has a growth defect at higher temperatures and on alternative carbon 
sources, and changes the expression of about 400 genes. 
Last, they demonstrate that the Taf14-ET can undergo phase separation, and that phase 
separation is stimulated by EBM peptides. 
They demonstrate that EBM binding and phase separation is conserved for YEATS but not Bromo 
associated ET domains, across species. 

The experiments are mostly very clean and logical, and I would in principle recommend 
publication. 

However, I have a couple of technical and stylistic comments 

Major points: 
(1) I was wondering about the structural integrity of the Taf14 mutants in the ITC experiments 
(FIgure 2D, line 172 etc), arginine might be quite disruptive here.  Could the authors present 
some kind of data supporting addressing this point, for example gel filtration profiles or NRM, or 
alternatively use something smaller (Alanine or Serine) or maybe find a ‘rescuing’ mutant on the 
Sth1 side? 
(2) For the in vivo experiments, the authors work with the Sth1-M4 mutation, and claim that this 
disrupts specifically the Taf14 interaction. However, this could also affect other proteins (ET 
domains?) binding to the same motif.  Also, could the authors please quantify the WB in Figure 
2E ? It looks as if the M4 mutant was already a bit less in the input. 
(3) To be better able to judge the quality of the data, it would be essential to show the original ITC 
curves for the experiments in Figure 4A, not only the final Kd values; only some of them are 
shown in Figure 4C/D 
(4) For the phase separation (LLPS) assays, the authors should provide more controls.  My first 
concern is the Taf14 protein they use - does it still carry the GST tag, and what GFP variant are 
they using? ‘Canonical’ GFP tends to dimerise, and can undergo phase separation by itself, 
especially in strongly crowding conditions like 10% PEG.  Second, I am wondering about the 
addition of the EBM factors; this could also be additional protein crowding. As a control, they 
should use RFP only (and whatever else is attached to these peptides) 

Minor points: 
(1) In the introduction, it might be nice to introduce Sth1 with a sentence or two, since it will play 
a major role in this paper 
(2) In Supplementary Figure 2A, is it possible that the labelling of the ITC curves (for I1208A and 
L1210A) was mixed up? 
(3) For gene expression analysis, the authors use RNA sequencing and see differences when 
comparing the transcriptome of the wild-type versus the Sth1-M4 carrying strain. The experiments 
performed seem valid to me. I am aware that this might go beyond the scope of this manuscript, 
but the ultimate proof of their model would be to demonstrate that Taf14 or RSC recruitment is 
affected at the genes where they see changes. 
(4) See major comment 4: a list of plasmids / exprssion constructs would be useful 
(5) I find the structure in Figure 2C a bit hard to read, especially which amino acids interact. 
Maybe they can find a clearer perspective, or show two alternative ones ? 
(6) In Figure 6E, a scale bar would be helpful. 
(7) At some selected points, the English grammar was not yet perfect, it might be good to send it 
to a native speaker for a quick read-through. 

Reviewer #4 (Remarks to the Author): 

This manuscript reported that the extra-terminal domain of Taf14 recognizes a common motif in 
multiple transcriptional coactivator proteins from diverse nuclear complexes, and the authors 
demonstrate that Taf14ET could form liquid-liquid phase separation (LLPS) in vitro. This 
manuscript not only identifying the molecular mechanism of Taf14ET-related transcriptional 
mechanisms, but helps us to understand the diverse cellular processes that Taf14ET regulating. 
However, the functions of Taf14-Sth1 interaction have not fully studied. I believe it is important for 
this manuscript, this is alsl why we investigate the transcriptional machineries of Taf14. So, some 
improvements might need to take into consideration before publication. In this case, the decision 
of this manuscript is major revision. 
Some comments and suggestions: 
Major comments: 
1. Why the authors use ‘Sth1’ as an example? I suggest the author explain the reason in 
Introduction section. 
2. Line 116: In figure 1B, the authors mentioned ‘GST-Taf14174-244 could pull-down Sth11183-
1359, Sth11183-1240, and 587 Sth11199-1225, but not Sth11248-1359.’ But in Line 116, the 
author reported that ‘the Sth1 fragments comprising residues 1183-1240 and 1199-1225 were 
able to associate with Taf14ET’. Why the Sth11183-1359 is lost? 
3. In Line 120, the authors mentioned that ‘the interaction between Taf14ET and Sth11183-1240 
is dominated by hydrophobic interactions rather than electrostatic contacts’, but I do not know 
why the authors indicated that ‘Electrostatic interactions further stabilize the Sth1EBMC-Taf14ET 
interaction.’? So, the interaction was dominated by hydrophobic and electrostatic interactions? 
4. Line 139: If we deleted the 9-residue core fragment, and determined the binding capacity? If 
so, we can further confirm that this motif directly interacts with Taf14ET through mutagenesis 
study. 
5. Why the authors choose temperatures as the phenotype’s experiments? I suggest the authors 
choose different types of stress conditions to confirm the universality of the conclusion. Such as 
various pH, osmotic pressure. In Figure 3A, the authors did not study the phenotype at 32 oC, how 
to ensure that the strain grew at 32 oC and below? Figure 3B, how to determine the appropriate 
concentration of the DNA-damage stress conditions? It is difficult to displayed that no sensitivity to 
MMS was detected in figure 3B. The author is advised to adjust the contrast of the figure or find a 
suitable dilution gradient. 
6. Line 219: Did the authors select 7 genes for verification? If so, based on what? In addition, the 
expression levels of these genes in the RNA-seq experiments should also be marked in Figure 3D. 
7. Line 227: I do not think the authors have well studied the metabolic pathways that Taf14-Sth1 
interaction participated. 
8. Line 239: where are the results of Taf2? I have not found the corresponding figure. 

Minor comments: 
1. Line 63: ‘Saccharomyces cerevisiae’ changed to ‘S. cerevisiae’, please check other expression. 
2. Line 93: as an example 
3. Line 98: ‘analysis’ to ‘analyses’. 
4. Line 119: dissociation constant (Kd); ‘55 nM’ 
5. Line 125: ‘nuclear magnetic resonance (NMR)’ Abbreviations should be marked when they first 
appear. In addition, the author needs to check other abbreviations. Some abbreviations need to be 
marked with their full names. 

6. Line 201: ‘sth1’ 
7. Line 227: there is no figure 3H, is it figure 3F? 
8. Line 261: In Supplementary 4D, is there any control group? 
9. Line 287: ‘Taf14ETs’ 
10. Line 336: Please marked the input and output in figure 6D.