Peer review of: Interaction of the pioneer transcription factor GATA3 with nucleosomes (2020)

Reviewers' comments: 
Reviewer #1 (Remarks to the Author): 

In this article the authors follow up on earlier published observations of the GATA3 transcription 
having three distinct types of binding sites as first revealed by measuring accessibility using ATAC-
seq and coupled with GATA3 ChIP-seq. They expand upon this by doing capture MNase-seq to 
more precisely determine the nucleosome position in these three categories of GATA3 binding sites 
and validate the earlier observations in that GATA3 either binds to a site already outside of the 
nucleosome bound region or within a site that is initially bound by a nucleosome that either causes 
the site to move outside of the nucleosome or to stay within the nucleosome after binding. They 
find for the nucleosome bound GATA3 that in the two subclasses they have different locations they 
prefer to bind in the nucleosome. The authors provide biochemical evidence for GATA3 working as 
pioneer transcription using purified GATA3 and mononucleosomes reconstituted on 601 DNA. They 
engineered GATA3 binding sites into different positions within the 601 DNA focusing on the SHL5 
and SHL2 positions like that observed in vivo and measured the binding affinity of GATA3 for these 
sites when assembled into nucleosomes. These experiments appear to have been well done, 
except that in Figure 2 there is not mention of these experiments having been repeated and there 
is no additional data showing how reproducible these results are. Similar data is also shown in 
Figure 4 with two replicates mentioned and the other replicate being shown in Supplementary 
Figure 4 (not Supplementary Figure 3). Unfortunately, the replicate shown does not give the same 
results as in Figure 4 and instead in Supplementary Figure 4 GATA3 overall appears to have a 
higher affinity for nucleosomes. There needs to be more replicates performed and the data 
quantitatively analyzed to confirm the statistical reproducibility of the data in both Figures 2 and 4. 
It was not possible to effectively compare the differences in GATA3 binding affinity as its site was 
varied without more repetition and quantitation being done. The fact that they still have GATA3 
binding to 601 nucleosomes when all of the GATA3 sites gone is disconcerting and not well 
addressed in the manuscript. Although it doesn’t form a specific band, the general smear 
nonetheless indicates that GATA3 is binding but not at a specific site. Why does GATA3 bind so 
well nonspecifically to nucleosomes with an affinity approximately comparable to that with 
nucleosomes containing GATA3 sites? It wasn’t clear in the experiments trying to show that GATA3 
prefers to bind to the SHL5 position that the GATA3 binding site at SHL+3 wasn’t removed to 
ensure that it wasn’t also contributing. 

Using cryo-EM the authors were able to solve the structure of GATA3 bound primarily to the SHL5 
and secondarily to the SHL6 positions of nucleosomes to 3.15 angstroms. It was valuable to see 
this structure and does provide additional insight. Given that GATA3 can also bind at SHL2 it would 
have been particularly important if they could have solved this structure particularly because 
GATA3 binding at this position is more likely to perturb the nucleosome than at SHL5 and SHL6. 
The specific requirements for the spacing between the GAT sites was informative and the different 
spacing requirement depending on the orientation of the two half sites. I found the statement 
about the link between chromatin remodelers that bind at SHL2 and GATA3 binding at this same 
location. Am I correct in think that rather than a link or synergy between them wouldn’t this 
represent more of a competition for the same binding site? 

Minor issues 
(1) Line 140 “SHL25b” should probably be SHL5b. 
(2) In Figure 1F the authors did not explain the differences between ellipses with a dashed versus 
solid line. 
(3) I was wondering why there is a loss of signal at the highest concentration of GATA3 in the gel 
shift assays? 
(4) Line 667 should read Supplementary Figure 4 and not 3. 
(5) The gel image in Supplementary Figure 2 looked quite skewed or distorted and was wondering 
why. 


Reviewer #2 (Remarks to the Author): 

In this study, Tanaka et al purports to study the interaction between GATA3 and nucleosomes. 
They first defined nucleosomal profiles at GATA3 binding sites before and after GATA3 binding in a 
system where this factor elicits its pioneer action. In contrast to accessible sites (G2) that are 
depleted in nucleosomes, both sites that are permissive for pioneer action (G1) and sites that are 
remodelling-resistant (G3) have nucleosomes at the binding sites before GATA3 binding. The G1 
and G3 subgroups present with different organizations of GAT motifs relative to the ChIPSeq 
GATA3 peak center. Using the purified GATA3 zinc finger DNA binding domain, the authors 
characterized its interaction with reconstituted nucleosomes and found the strongest binding with 
sites enriched in the remodelling-resistant G3 subset. They used cryo-EM to define the structure of 
this complex and mutagenesis to validate its interpretation. They finally went back to their GATA3 
ChIPseq data to identify GAT motifs enriched around the peaks of the G1-G3 subsets. In 
agreement with the in vitro nucleosome interaction data, they found enrichment of the direct 
repeat separated by 6bp in the G3 group but more strikingly observed that palindromic sites 
separated by 3 or 4 bp are greatly enriched in the G1 pioneer and G2/G3 groups. 

Although very interesting, this is an odd paper that finishes where it should have started, namely 
analysis of GAT sequences present under GATA3 peaks! Indeed, the most striking observation 
presented in Fig.5 is enrichment of the palindromic sites at the pioneer-permissive sites. Hence, 
these sites appear the most interesting in terms of understanding the GATA3 pioneer action. 
However, the authors did not investigate this in any way! The palindromic nature of these sites 
suggests dimer interaction and prior crystallography data involving some of the same authors 
showed GATA3 dimer interaction with DNA, albeit with different spacing between the palindrome 
half-sites. That makes the observation of the 3/4 bp-spaced palindromes under the GATA3 peaks 
even more provocative as it suggests that such spacing might be critical for pioneer ability. 

Nonetheless, the authors have adequately documented the structure of GATA3 bound at the edge 
of nucleosomes as present in the remodelling-resistant G3 subset. In summary, this reviewer 
cannot escape but think that this is an incomplete study that misses the most important aspect of 
GATA3 interaction with pioneered sites within nucleosomes. It is thus preliminary in this reviewer’s 
opinion. 

Specific comments 

P.7L.193 Is it really Table 2 that should be referenced or rather Supplementary Table S1? 

The Discussion is rather superficial and in good part a rehash of the Results section. None of the 
points raised above in this review are approached. It would seem natural to extensively discuss 
the finding of the GAT sequences found under the GATA3 peaks in the discussion of a paper that 
purports to define the interaction of this pioneer with nucleosomes. 



Reviewer #3 (Remarks to the Author): 

In this study Tanaka and collegues describe the interaction between the pioneer transcription 
factor GATA3 and nucleosomes. The author use high-res nucleosome mapping in human cells to 
study the relation between the position of GATA motifs on the NCP surface and productive 
enhancer formation. They find that enhancer formation is productive when GATA binding sites map 
in proximity of the dyad axis. The authors use Widom601 nucleosomes containing a GATA binding 
site to identify biochemically tractable GATA3-nucleosome complexes for cryo-EM analysis. A 
3.15A res cryo-EM structure is reported, which contains nucleosome and the GATA3 zinc finger 
motifs, binding on consecutive major grooves. No significant structural change in the histone 
octamer or the nucleosomal DNA structure is reported. The authors conclude that productive new 
enchancer formation might derive from specific spacing of GATA3 recongition sequences on the 
nucleosomal DNA. 

This is a very thorough study and the cryo-EM work is state of the art. However, in my opinion, 
two issues should be addressed before this study can be accepted for publication. 

1. The authors should explain why a 145 bp Widom 601 positioning sequence was selected for 
biochemical and cryo-EM characterisation. Has the use of a natural nucleosomal DNA sequence 
containing GATA3 binding sites been attempted - if so what were the hurdles encountered / if not, 
how can the author justify their choice? 

2. Related to the first point. Could there be additional DNA engagement modes for GATA3 on 
nucleosomal DNA (involving DNA distortions), which are not visible because of the use of the 
strong positioning Widom601 sequence? This point could be addressed by the authors when, on 
PAGE 10 line 258-260, they claim: "The structure revealed that the GATA 3 binding did not induce 
a structural change of the histone core and the DNA path in the nucleosome". 

I should stress that none of these are serious concerns, although I believe clarifying these points 
in the text will be appropriate. 

MINOR POINTS 

3. Have the authors attempted to characterise the complex in the absence of a crosslinking agent 
and what was the outcome? 

4. What is the estimated concentration for the protein sample used for cryo-EM grid preparation 
(Line 405 on page 14). 

5. It would be good to complement FIgure 3B with a supplementary FIgure comparing the 
structure of GATA3znf on nucleosomal DNA (this cryoEM study), with the crystal structure of the 
complex with naked DNA.