Peer review of: LSD1-mediated enhancer silencing attenuates retinoic acid signalling during pancreatic (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The work by Vinckier et al. examines the role of LDS1 in enhancer decommissioning during 
endodermal (gut/pancreas) embryonic development. The authors show that LSD1 binding to 
enhancers induces demethylation of H3K4 and promotes enhancer decommissioning. In this way, 
LSD1-mediated enhancer silencing acts as an epigenetic intrinsic mechanism to switch off the 
transcriptional response to an inductive developmental signal. This mechanism could explain how 
signaling cues are interpreted in developing tissues and how they are temporally limited to defined 
windows of competence for developmental transitions. 
 
The authors use hESC and mouse knockouts to show that early inhibition or deletion of LSD1 
abolish the later production of endocrine pancreatic cells. Lack of LSD1 results in persistence of 
H3K4me1 and me2 in enhancers that remained poised for activation. Genomic analysis of 
activated genes in LSD1 early inhibition (G1 group at enhancers) indicated the involvement of 
retinoic acid signaling. Persistence of RA signaling upon early LSD1 inhibition seems to prevent 
further endocrine differentiation. 
 
The manuscript is very well written and data are clearly explained. The questions are highly 
relevant to a wide audience of developmental biologists and provide a framework to understand 
longstanding questions in the field. There are however a few criticisms and suggestions that should 
be addressed. 
 
 
Major points 
 
1. The B27 supplement the authors use in their experiments usually contains retinoic acid 
(RA)/vitaminA at variable concentrations, usually around 1ug/ml. Can the authors please clarify 
whether the B27 they use in their culture at days 8-12 contains retinoic acid or is without it? If it 
contains RA, can the authors perhaps rephrase the RA extension/persistence concept with the 
more appropriate dampening of response to RA, which is also supported by the data using RA late 
activation after early LSD1 inhibition? 
 
2. N number is equal 2 for most experiments. We understand that the phenotype is quite clear and 
strong, so it might not need a third experiment to reach significance, but it would be good to know 
that the authors have repeated it multiple times and they are confident about the (non) variability 
of their assays (eg hESC differentiation protocol, area of the embryonic epithelium?)? 
 
3. In figure 5, if the area of the epithelium is not changed in LSD1 knockouts and yet the 
endocrine cells/islets (which should be 2-6%) are lost, is there any fate switch? What happens to 
the cells that do not differentiate to the endocrine lineage? Do they remain stuck at an earlier 
stage? The authors could look at the expression Ngn3 to comment on where in the lineage cells 
remain stuck. 
 
4. What about the role of LSD1 on G2 and G3 groups? For example, G3 group has less acetylation 
already in PP1, which is a prerequisite for LSD1 demethylase activity, and yet bound LSD1 does 
not remove methyl groups (methylation is actually increased)? Why? 
 
5. Figure 4 looks at upregulated genes and their relationship to RA signaling. If similar analysis is 
done with the down-regulated genes, is no relationship observed? 
 
6. RA late treatment seems to give rise to multihormonal cells (fig 5b)? This deserves comment. 
The authors could try to treat pancreatic explants with RA early and late and look at endocrine 
cells to see both whether they can recapitulate LDS1 knockout data but also whether RA treatment 

at later stages results in polyhormonal cells. This could be really important for understanding the 
mechanisms that segregate endocrine subtypes and might be relevant for the generation of mono-
hormonal insulin-producing beta cells for diabetes. 
 
7. Fig 6f: why do you have so many LSD1 positive cells in LSD1Delta-early (around but also far 
from insulin+ cells)? Is this picture representative? 
 
 
Minor points 
 
1. It would be helpful to add in figure 1 the days to reach the different intermediates 
 
2. Is LSD1 ubiquitous? What about other tissues in which RA plays an important and biphasic role? 
For ex in the nervous system, RA is first involved in posteriorization and then it is essential for 
neuronal differentiation. The authors should add something about this in the discussion. 
 
3. If RXR ChIpseq has been undertaken and is referred to, the data about location of peaks etc. 
should be made available. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
 
NCOMMS-18-30737 - LSD1-mediated enhancer silencing enables endocrine cell development 
through attenuation of retinoic acid signalling 
Nicholas K. Vinckier, Nisha A. Patel , Allen Wang , Jinzhao Wang , Hung-Ping Shih, Jianxun Wang , 
Ulupi S. Jhala , Michael G. Rosenfeld , Christopher W. Benner and Maike Sander 
 
In this manuscript the authors, using a well-defined stepwise differentiation program of hECS into 
pancreatic endocrine cells along with relevant genomic approaches, suggest that LSD1 affects the 
epigenetics landscape of the differentiation program in a define window and demonstrate that 
LSD1-mediated enhancer decommissioning affects retinoic acid (RA)-induces expression. The 
authors also show that, in the absence of LSD1, RA-depending enhancers remain active despite 
the absence of RA, with a consequent failure to progress into the differentiation program. 
Collectively, their findings indicate that LSD1 functions at a defined window of the differentiation 
program, and loss of LSD1 alters epigenetics landscape affecting RA signaling. 
The findings reported are strongly indicative of the role of LSD1 in the appropriate control of 
endocrine program and highlight a novel time-dependent function of LSD1 in endocrine 
differentiation. I share with the authors the possibility that their findings might be broadly 
relevant. 
 
Overall, the authors have made potentially interesting findings but, some data presented in the MS 
are over-interpreted and there are several areas where it falls somewhat sort. Some additional 
experiments as well as a more accurate presentation of the data will definitively improve the 
significance of their observations. 
 
Specific comments 
 
1. A compounding issue is that the authors consider the use of drugs such as the irreversible TCP 
and the reversible SP2509 (along with GSK-LSD1 and GSK2879552) equivalent to LSD1 knock-out 
and this is not fully correct. These drugs, especially TCP (used to generate most of the data), also 
inhibits the activity of others proteins. This point raises the question of the selectivity of the LSD1 
inhibitors since TCP in addition to inhibit monoamine oxidases MAOA/B, also affects the LSD1 
paralog LSD2. I appreciate the in vivo data presented in Figure 6 supporting the specific role of 

LSD1 in endocrine cell formation, however, they are insufficient to validate all data obtained in the 
in vitro studies using pharmacological inhibition of LSD1. It is pertinent to consider that like LSD1, 
LSD2/KDM1B is a FAD-dependent enzyme histone demethylase and it participates to gene 
transcription at least in part through control of transcription elongation control. I would urge the 
authors to determine the specificity of LSD1 inhibition at PP1-PP2 stage by CRISPR-Cas9 editing 
(or similar specific LSD1 genetic ablation). Alternatively, if LSD2 is expressed in hESCs, (likely to 
be the case), LSD2 Chip-seqs in the presence and absence of TCP would help to segregate the 
relative contribution of both KDM1A and KDM1B at PP1 to PP2 transition. 
 
2. ChIP-seqs for LSD1 were carried out at PP1 stage with or without LSD1 inhibitor (I assume 
TCP??). For how long the cells were treated with TCP before chromatin isolation? Specifically, I 
wonder whether the chromatins from treated and untreated cells were prepared at the same time 
of differentiation protocol. As it stands the LSD1 inhibitor protocol is unclear. 
 
3. Previous work in mouse ESCs (Whyte et al. 2012) reported LSD1 signals at core promoter 
regions. In addition, several reports indicate the presence of LSD1 signals at TSS. From the 
presented data it is not clear how many of the 15,084 LSD1 peaks are at TSS? And….. if there are 
examples where LSD1 is present in both distal and core TSS, or only at distal, or only at TSS. 
Please provide these informations. 
 
4. The authors state that they:…… isolated enhancers that are active at PP1 and/or PP2 and also 
bound by LSD1 at PP1, ….These putative enhancers were divided in 3 groups G1, G2 and G3, on 
the basis of the relative abundance of H3K27ac. I assume that the authors imply that the distal 
LSD1-bound regions (enhancers) are also co-occupy by hESC master transcription factors such 
Oct4, Sox2, Myc and Nanog and the Mediator coactivator. I reckon that this assumption must be 
validated. It will be sufficient to demonstrate by ChIP data that a specific distal LSD1 binding 
region (such as HOXA1 shown in Fig 2 E) is also co-occupy by a dedicate TF or Mediator factor. 
 
5. LSD1 is a FAD-dependent enzyme. Is the FAD level constant throughout the hECS 
differentiation program toward pancreatic endocrine cell lineage? The rate-limiting role for FAD in 
LSD1-mediated effects has been recently documented by Yang et al. EMBO J. 2017, 36: 1011–
1028, doi:10.15252/embj.201694408. Reduction of FAD levels at PP2 stage will provide a plausible 
explanation of the temporal window of LSD1 function. 
 
6. In fig 1 the effects of LSD1 (both early and late) at EN stage is revealed only by flow cytometry. 
I suggest that the authors validate the phenotype also by qRT-PCR, as shown in panel A of 
Supplementary Fig 5. 
 
7. Provide qRT-PCR of LSD1 or WB in Supp. 1a (no FPKM). It is not clear if the LSD1 wave of 
expression (low in DE, high in GT) is significative. Similarly, data from Fig. 4 panel H, and 
Supplementary Fig 6 E, should be validated by qRT-PCR. 
 
8. Legend Supp. Fig. 3. CYP26A1 is duplicated. 
 
9. To provide sufficient details for the work to be reproduced successfully, the authors should add 
details of the pharmacological conditions used. 
 
10. The description of figures is often rather cryptic. The figure legends need to be specified so 
that one can understand what was done. As it stands too much guessing is necessary. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
This manuscript provides evidence that the histone demethylase LSD1 regulates the epigenetic 

status of developmental enhancers to control how those enhancers respond to extracellular 
signals, namely retinoic acid during pancreas specification. While LSD1 is known to 
“decommission” enhancers in the context of cell signaling the current study suggests that this 
provides a mechanistic basis for developmental competence and how the duration of 
transcriptional responses to extracellular signals are modulated and reset during lineage 
progression. This is an important area of work that in principle the findings represents an 
important advance in the field. However, there are a few important issues that need to be 
addressed to support the model. 
 
1. What recruits LDS1 to enhancers and when is it first there. If the model is correct then LSD1 
should not be at G1 enhancers at the gut tube stage. If it is at there, then it should prevent those 
enhancers from becoming active in response to RA signaling. The authors really need to show 
LSD1 binding at the GT-stage to address this point as is it a key aspect of the model. 
 
2. The idea that LSD1 regulates competence of an enhancer to respond to RA-RxR activity is 
attractive, but the data that the authors show is mostly correlative. If the model is correct then 
ectopic LSD1 should prevent (or attenuate) RA from activating these enhancers. 
 
3. The data and interpretation in Fig. 2 d-f is confusing. The model proposes that LSD1 
demethylates after the enhancer after RA is withdrawn from the culture and that in the LDS1i 
demethylation doesn’t happen and RA-responsive genes expression persists. But if this is true then 
I would expect an increase in K27ac in the LSD1i to accompany the persistent gene expression. 
How do you get persistent expression in the absence of K27Ac? 
 
 
4. The evidence that the modest ~2-fold increase in me2 on G1 peaks can account for the 
persistent expression is not very strong. These two observations might be true-true and unrelated. 
An alternative explanation is that LSD1 is doing something else, independent of K4me2 levels to 
regulate expression. 
 
5. In Fig. 4h it would be good to also show the expression of these key LSD1/RA-responsive genes 
at PP1 so that the reader has a clear understanding of what this elevated expression at PP2 is 
relative to that see normally at the PP1 stage. The elevation could be very modest only 10% of 
PP1 levels. Fig. 3d is a good stat but the authors should show the expression of the key LSD1/RA-
targets. Similarly in Fig. 5 it would be good to show the RNA-seq/RT-PCR data in a histogram to 
more clearly show the expression of key LSD1/RA-responsive genes at PP1, PP2 and EN +/-late RA 
to be able to compare the level of expression. Are the LSD1/RA-regulated genes really not 
activated at all when RA is added late (it is hard to extract that info from the current figure). When 
they are activated in the LSD1i+RAlate condition what is the level of expression relative to that 
normally seen at PP1. 
 
6. If the LSD1i or RA extended cells they are stuck at a progenitor state then PCA analysis of the 
RNA-seq might show that the PP2-LSD1i or PP2-extended RA are more similar to PP1 cells. 
 
7. For the mouse genetics any evidence of elevated RA-target gene (Hox) expression in the LSD1 
KO? 
 
 
 
Minor points 
1. Need to report the antibodies used for ChIP 
 
2. While the enrichment analysis of motifs is okay, the authors should show the motif analysis of 
LSD1 ChIP peaks from G2, G3 and the rest of the genome other than G1-3. Perhaps RxR is just a 
common feature of all of these. 

 
 
3. The authors definition of decommissioned and deactivated enhancers changes a bit at different 
points in the paper. G1 enhancers are selected as “decommissioned” based on loss of K27ac. But 
then they classify decommissioned based on the removal of me2, in the context of no change in 
K27ac. 
 
 
 
Reviewer #4: 
Remarks to the Author: 
Summary: 
In this manuscript the authors first show that endocrine cell development in differentiating hESC 
cultures requires LSD1 activity during a limited time window when into the first recognizable 
pancreas progenitors (PP1 cells) differentiate into late pancreas progenitors (PP2 cells), but not 
during differentiation of PP2 cells into the endocrine lineage (EN cells). 
The authors then conducted LSD1 ChIP-seq to identify potential target genes. Analyses of the 
LSD1 ChIP-seq dataset together with previously published ChIP-seq data sets for H3K27Ac and 
H3K4 Me1/2 identified an LSD1-regulated set of enhancers (G1) that is activated when gut tube 
(GT) progenitors evolve into PP1 cells and deactivated (deacetylated) and decommissioned 
(demethylated) when these factors are withdrawn during the PP1 to transition PP2. The authors 
found that deacetylation of these enhancers occurred largely independent of LSD1, but that LSD1 
was required for demethylation. Comparison to RNA-seq data from LSD1-inhibited cells suggested 
that genes up-regulated after LSD1 inhibition were direct LSD1 target genes, whereas down-
regulated genes were not directly regulated by LSD1. The authors then found an enrichment for 
genes involved in RA signaling among G1 enhancers. 
Transcription factor binding motif enrichment analysis showed significant enrichment of the motif 
for the RAR/RXR heterodimer at G1 enhancers and RXR ChIP-seq analysis indicated binding of 
RAR/RXR receptors to about one third of the G1 enhancers. The authors then show that extending 
the RA exposure into the PP1 to PP2 transition mimicked the phenotype seen upon LSD1 inhibition, 
while progenitor cell markers were unaffected. RNA-seq analysis was then used to identify genes 
dysregulated as a result of prolonged RA exposure and genes up-regulated by LSD1 inhibition 
were enriched among the genes also up-regulated after extended RA exposure. Similar to the 
LSD1 inhibition, this effect of prolonged RA treatment was not seen at the later PP2 to EN 
transition. Interestingly, prior LSD1 inhibition led to enhanced induction of RA responsive genes, 
suggesting that LSD1 dampens future RA responsiveness in cells that experienced prior RA 
exposure. 
Lastly, the authors demonstrate that pancreatic Lsd1 deletion in vivo in mice results in a strong 
reduction in endocrine cells and that this Lsd1 dependency is restricted to a narrow time window 
between E10.5 and E12.5. 
 
Critique: 
This is a clearly written paper with some interesting and novel findings. Generally, the experiments 
are well performed and many of the conclusions drawn are justified. However, the main conclusion 
is not supported by the data in their present form. Thus, I would like to see the following major 
issues addressed before recommending publication: 
 
1) The motif search done by the authors, which revealed the RA-response element being enriched 
is not particularly impressive given that Table S6 shows that only 1.14% of the LSD1 target 
sequences had this motif. Adding the DR1 motif (line 20 in Table S6) at 2.4% only slightly 
improves this. It is thus surprising that nearly one third of the G1 enhancers showed RXR binding, 
but without access to the ChIP-seq data, one cannot assess the significance of this observation. 
The GEO data set should therefore be made available to the reviewers. 
 
2) The phenotypic similarity between LSD1 inhibition and prolonged RA exposure is, albeit 

interesting, mostly correlative in nature and none of the experiments directly test whether the 
effect of LSD1 inhibition is solely caused by a failure to decommission RA-responsive enhancers 
and thus prolonged activity of the genes regulated by those enhancers. 
Thus, the link between LSD1 and RA signaling is somewhat tenuous. To bolster their conclusions 
the authors should test whether inhibition of RA signaling, for example with an inverse agonist that 
prevents activation even from “open” enhancers, can rescue the effect of LSD1 inhibition. This 
should be done both in the hESC cultures and in explants from Lsd1-deficient embryonic mouse 
pancreas. 
 
3) Endocrine cells differentiate from pancreatic progenitor cells via a NEUROG3 expressing 
precursor stage, yet nothing is mentioned about this in the paper. In the discussion the authors 
refer to “the endocrine-specified pancreatic progenitor cell stage” but it is unclear what they mean 
by this. Do the authors mean NEUROG3+ cells here? 
More importantly, the authors make no attempt to decipher whether the effect of LSD1 inhibition 
or extended RA treatment on EN cell differentiation is executed by preventing induction of NGN3 or 
preventing the differentiation of NGN3+ cells into EN cells. The tools for making this distinction are 
readily available and have previously been used by the authors. This is of major importance for the 
field and for the usefulness of these findings in the translation to hESC differentiation protocols. 
Thus, this omission should be remedied. 
 
Minor issues: 
 
Antibodies used for ChIP-seq are not listed in the methods section.