Peer review of: Global translation during early development depends on the essential transcription factor (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this article the authors describe and characterise the role of Prdm10 in early murine development, 
both in vivo and in vitro. They found that homozygous embryos are lethal post implantation and E4.5 
embryos demonstrate a growth phenotype. To better understand the function of Prdm10 in early 
development the authors derived mouse embryonic stem cells (mESCs) from the their conditional 
Prdm10 allele and found that mutant ESCs have growth defects while the expression of pluripotency 
markers is only modestly affected, indicating that the gene is essential for normal growth, but not for 
maintenance of the pluripotent state. The authors identify Prdm10 targets by ChIP Seq and surprisingly 
find its binding restricted to the promoter region of annotated genes. RNA-Seq following Prdm10 knock 
out, alongside their ChIPseq, demonstrated a role for Prdm10 in regulating translation. They then focus 
on one downstream target, Eif3b, a component of the eIF3 complex that is involved in translation 
initiation. Knockdown of Eif3b results in severe growth defects that are rescued by over expressing a 
shRNA-resistant Eif3b. Moreover, they show that overexpression of Eif3b in the Prdm10 null cells gives 
partial rescue. Finally, the authors do polysome profiling in the Prdm10 mutant cells and show a 
reduction in translation rates. 

The notion that translation can regulate cell state in development would be of fundamental interest. 
However, the idea that a single new transcription factor activates the transcription of translational 
regulators, is not particularly novel. If Prdm10 had a lineage specific phenotype then this manuscript 
might have more general interest and in particular, if they could directly link the regulation of 
translation to that phenotype, this would be an important and exciting observation. The immune 
fluorescence in figure S1E and F, could suggest a defect specifically in the ICM, but there is no relative 
quantification. In addition, the phenotype in ESCs in never really explored, while they show relatively 
little change in Nanog, KLF2, Oct4 and Esrrb there is no real analysis of the phenotype and or 
differentiation competence of these cells. It is not clear from the current manuscript whether they can 
passage mutant cells and under what culture conditions they could do this (i.e could they survive in 
2i/LIF or other alternative defined culture medias). Based on what is in the paper, this factor could be a 
general regulator of translation that is expressed in ESCs, explaining why it was pulled out of multiple 
gene trap screens, but it is hard to see how it is related to development and differentiation. 

Specific Comments: 
1. The data in this paper is scientifically and technically sound however the authors should improve the 
characterisation of Prdm10 null mESCs phenotype (as discussed above). The authors conclude that 
Prdm10 affects cell growth by analysing doubling times, colony formation and morphology. Cell cycle 
analyses (especially since GO terms associated with cell cycle are also enriched in the differential 
expression analysis), staining for proliferation markers (like Ki67) would strengthen the claims that 
Prdm10 is affecting cell growth/proliferation. Additionally, the authors could also investigate cell death 
(caspase 3 staining) in Prdm10 null cells. 
2. The impact of the Prdm10 KO on pluripotency should also be better assessed (as discussed above). 

Also details of specific experiments were not clear. In Fig. S2i,j, how many days post-induction was SSEA-
1 staining and AP staining done? In Fig. S2j, only one colony is shown for the AP staining, with no 
quantification. In Fig. S2h, although not significant, the expression of pluripotent genes seems slightly 
affected by Prdm10 KO. This was three to five days following the Prdm10 knock out, but they only see 
significant changes in the doubling time of these cells after day five. What is the expression of these 
genes at the time points where they observe a pronounced phenotype? 
3. Although the authors show that Prdm10 leads to a decrease in translation rates that are rescued by 
Eif3b expression, the Prdm10 phenotype is only partially rescued. What else accounts for the Prdm10 
phenotype? 
4. Are there other ribosomal proteins whose is regulated by Prdm10 or is it only Eif3b. In particular their 
GO analysis suggests translation regulation is important, so Prdm10 must be regulating other translation 
factors. 
4. The authors claim that Prdm10 regulates global translation. However, are their mRNA classes that are 
not affect by Prdm10 KO. If you completely block translation, then one would expect that the cells 
would not be able to divide at all. 
5. In both the embryonic and ESC phenotype, the authors should look at differentiation markers as well 
as Epiblast. 
6. In Prdm10 knock outs (Fig S3A) they still observe Prdm10 ChIP seq peaks, do they remove them from 
their analysis. 
7. In Fig. 2b the label of the Y axis be % of peaks, not peak counts? 
8. In Fig. 2E they have a red line for the controls, but never specify what their control is. 
9. It is very surprising that they mostly observe binding of this transcription factor at promoters, Why 
are there no intragenic or enhancer peaks? Is this for a technical reason? 

Reviewer #2 (Remarks to the Author): 

In this manuscript entitled “Global translation during early development depends on the essential 
transcription factor PRDM10”, Han et al. document an essential role for PRDM10, an uncharacterized 
zinc finger containing protein, in pre-implantation embryo and mESCs. PRDM10 apparently functions as 
a transcriptional activator. Han et al. undertake ChIP-seq and define a motif that responds to the 
presence of PRDM10. They also define the chang in transcriptome 2 and 4 days following ablation of 
PRDM10. Among the genes whose expression are regulated by PRDM10 is eIF3b. eIF3b is an essential 
gene so its suppression affects cell survival/proliferation. Ectopic expression of recombinant eIF3b 
partially rescues loss of PRDM10 (the rescue here is quite small – Figs 4f, g) indicating that other factors 
are also at play in mediating the PRDM10 lethality. They also show that PRDM10 is essential for normal 
growth of mESCs but is dispensable for maintenance of the pluripotent state. 
The only real weakness that I see in this paper is the link between PRDM10 essentially and effects on 
eIF3b expression and phenotype. It is not surprising that loss of eIF3b compromises cell growth as the 
gene has already been defined as essential. The question is how much of PRDM10’s effects is a 
consequence of eIF3b loss versus loss of expression of other targets. In fact, in terms of expression data, 

eIF3b ranks ~100th when the list of suppressed genes is rank ordered (Table S3). How many of these 
other genes, whose expression are more dramatically affected contribute to PRDM10’s essentiality? For 
example, I see eEF1D expression is affected more pronouncedly than eIF3b – why was this not pursued – 
it is also involved in translation? How many of these 100 mRNAs have PRDM10 binding sites in their 
regulatory regions? I think the link that PRDM10 regulates eIF3b is credible, the issue is how much of 
PDRM10’s effect is through eIF3b. 
Aside from this shortcoming, the paper is well written and the experiments appear to have been well 
performed. A few additional comments: 

p. 7, line 137. “contrast, PRDM10441-880 showed only weak affinity for the MUT probe in direct binding 
(Fig. 2g) and competition assays (Fig. 2h),”. I don’t think claims on affinity can be made here based on 
these experiments. If such claims are to be made, then Kd’s or Ka’s should be measured. 

p. 9 , line 176-179. I found the section that reads “Of these, we observed significant differences in 
expression (Padj < 0.05, fold-change > 2) for 52 and 76 genes at 2 and 4 days post-deletion 
respectively (Fig. 3c). Notably, the majority of genes bound and regulated by PRDM10 
(Padj < 0.05) showed decreased expression in Prdm10Δ/Δ mESCs (Fig. 3d).” confusing. I couldn’t 
understand how many of the genes whose expression are altered in the transcriptome expression data 
contain PRDM10 binding sites within their promoters. Is it 52 and 76 as stated or 228 – 263 as indicated 
in Fig 3d. 

Please label Y axis on Fig 4H. 

What proportion of PRDM10 is nuclear? Cell fractionation or immunofluorescence studies should be 
able to address this issue.