Peer review of: Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leu (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscrit by Deniz and co-workers the authors investigate if endogenous retroviruses 
serve as a source of enhancers in AML and how this may influence tumor heterogeneity and 
evolution. Overall, this is well written manuscript that is easy to understand and the message is 
clear. 

The study appears to be well designed and the results are nicely presented and interpreted. The 
choice of methodology is clearly state-of-the-art. My main concern with the manuscript is if these 
findings to are relevant to AML or if they primarily represent in vitro artefacts found in K562-cells? 
This concern somewhat dampens my enthusiasm with the manuscript and puts into question the 
potential impact of this study. 

Major comments 

1. It would be very valuable if the authors could strengthen the AML-dataset to make their 
observations less preliminary. Given the high heterogeneity of the AML-samples it is obvious that 
the study is underpowered. At least the authors should discuss this limitation more clearly. 

2.The title is overselling the results and should be toned down. The evidence that ERVs are 
oncogenic enhancers is rather weak. 

3.The CRISPRi experiment is interesting – but also difficult to fully interpret. Additional control 
experiments would be very valuable. For example, did the authors try the same experimental 
setup with a gRNA targeting a different ERV family of similar size that do not carry enhancer 
marks? It would also be very interesting to extend this experiment to a more relevant cell type, 
such as primary CD34+ cells. 

4.The CRISPR experiment in Fig 6 is also very interesting. However, additional controls would be 
very valuable. For example, what happens if they cut out a similar size piece of DNA just upstream 
or downstream of the ERV? 

In summary, I found this to be a very robust study that uses state-of-the-art technologies to study 
ERV biology. My only problem with the study is the weak link between the observations done in 
K562 cells and actual AML samples. 


Reviewer #2 (Remarks to the Author): 

Endogenous retroviral elements (ERVs), particularly their LTRs, have natural transcriptional 
regulatory capacity. While most of the several hundred thousand LTRs in the human genome are 
epigenetically repressed, some are active in certain normal contexts, providing promoters and 
enhancers to host genes. There has been increasing interest in determining if “activation” of 
normally repressed LTRs in cancer could lead to deregulation of genes and contribute to 
oncogenesis. As the authors point out, previous efforts have mainly focused on LTR promoter 
activity in cancer for mainly practical reasons. This study examines the role of ERV LTRs, 
particularly as enhancers, in AML, which is a heterogeneous disease with generally poor outcomes. 
They first search for potential enhancers in ERVs or other repeats by intersecting DNase 
hypersensitive sites (DHSs) in 32 AML samples (as generated by the Blueprint epigenome project) 
with Repeatmasker annotations. As normal controls they use data from CD34+ cells, macrophages 
and monocytes. They identify six families of ERV LTRs that are enriched for DHSs in some AML 
samples from two datasets and in a few AML cell lines compared to normal controls and call these 
A-DARs (for AML DHS-associated repeats). These A-DARs are associated with histone marks 
typical of enhancers and expression of nearby genes correlates with A-DAR chromatin status. They 
also looked at TF ChIP-seq data from the AML cell line K562 and identified over 200 TFs that bind 
at least 5% of LTRs of a given family, some of which are involved in hematopoiesis and AML. TF 
motif analysis revealed that, in most cases, presence or absence of the motif does not correlate 
with DHS status of a particular element. They then proceed with more functional experiments and 
show that deleting 3 select LTRs in AML cell lines with CRISPR leads to reduction of nearby gene 
expression. CRISPRi experiments targeting the LTR2 and 2B families in two AML cell lines showed 
some genes are downregulated and that cell proliferation was reduced in both cell lines. Finally, 
they focused on an LTR2 upstream of the APOC1 gene since APOC1 has previously been shown to 
maintain cell survival in AML. Deleting this LTR leads to a decrease in APOC1 expression and 
significant decreases in cell proliferation as judged by lack of survival of homozygous edited clones 
and increases in apoptosis. Examination of survival data in TCGA shows that high APOCI 
expression is correlated with poorer prognosis. 
Overall, this is a very well done study and the logic of the paper flows nicely. The methods are 
generally well described. It is a comprehensive body of work and adds significantly to the growing 
literature on the potential roles of ERVs in cancer. I just have some relatively minor concerns: 

1. This is not a concern but rather an complement. CRISPR deletion experiments showed that a 
particular LTR5B element acts as an enhancer of the nearby RPL7L1 gene. Instead of stopping 
there, the authors noted that this LTR has a SNP where the minor allele disrupts a MEFK binding 
site motif. Analysis of GTEX data showed that the minor allele is associated with lower RPL7L1 
expression in blood. While a minor point in the paper, it demonstrated to the field that 
investigators should consider sequence variation within LTRs, not just the presence of the LTR, 
when evaluating their regulatory effects – an aspect which has been greatly understudied to date. 

2. Please add a statement to confirm that the editing sites of the non-viable APOC1-LTR2 
homozygous clones were contained within the LTR2 and did not extend into the flanking 
sequences. Admittedly, it is unlikely that this happened but it would strengthen the argument. 

3. One limitation of a study like this that the authors should at least discuss is the common 
problem of declaring something “cancer-specific” when comparing data from a large set of cancer 
samples to data from a much smaller number of normal samples. Since many of their analyses 
require only that the epigenetic mark or promoter activity, etc. be present in at least two AML 
samples but not in the normal differentiated cells (monocytes and macrophages), how can the 
authors be confident that a larger collection of normal cell samples might also have a few which 
display that particular mark or activity? A related problem is determining which “normal” cells to 
compare to AML samples. Here the authors use data from monocytes and macrophages and one 
could argue that fully differentiated cells are not the most appropriate control to compare to a 
poorly differentiated blood cancer. Perhaps some of the differences in ERV epigenetic marks or 
promoter activity have nothing to do with the cancer but just to the degree of differentiation. In 
other words, one wonders what fraction represent true “activation” of an LTR which is normally 
never active versus cases where the LTR is active in some normal tissues or developmental stages 
but just not in the few normal samples that the authors used to compare to the AMLs. The authors 
should add some discussion acknowledging/addressing these issues. 

4. In the first figure (fig 1B) and associated text, five transposon families appear to be enriched for 
DHSs in all samples and were therefore eliminated from further analysis. Two of these (TAR1 and 
MSR1) are satellite repeats and U1 is a snoRNA. These are not transposons and should not have 
been included in the screening method in the first place. A Blat search shows that most copies of 
the MER57E3 LTR are in the first intron of ZNF genes, having amplified as part of ZNF gene 
duplications, so also is not the typical dispersed transposon and should probably also not even be 
shown. Its DHS enrichment could well be due to proximity to ZNF gene promoters. The last one, 
LTR13, is also somewhat strange as many copies seem to be located in regions that don’t even 
align with Rhesus (according to a brief search using BLAT). 

5. For the data in Figure 1E, which shows the upregulated genes associated with a DHS at a 
nearby ERV, please provide a supplementary table with the gene names, coordinates of the ERV 
and the distance of the ERV to the TSS of the gene. 

6. Related to the above data, the authors discuss three genes at the bottom of page 6 that have 
some link to AML prognosis. Please elaborate a little more. Is up or down regulation of these genes 
linked to worse outcome? 

7. In the section where the authors look for LTRs acting as promoters (page 7), while “promoter 
effects” seem less important than enhancer effects, At least one gene (RHEX) in the associated 
Table is probably worth mentioning. LTR-RHEX transcripts are present in a large fraction of AMLs 
and the LTR2B is one of two annotated promoters in RefSeq. Since this gene is “regulator of 
hemoglobinization and erythroid cell expansion”, it would be worth commenting on it since it 
shows up in such a large fraction of AMLs. 

8. Related to the above, please include a paragraph on how ab initio transcriptome assembly was 
performed. As well, it is stated in the methods that the assembled transcripts were “visually 
inspected”. Were these checked against other sources (eg. ESTs or Gencode or FANTOM5) for 
evidence that the LTRs are indeed the promoters? As an example, for the SUOX gene, the LTR12C 
seems to provide part of an internal exon in UCSC and is certainly not the promoter. 

9. Several of the quoted references are not in the reference list. Please fix. 

Reviewed by Dixie Mager 



Reviewer #3 (Remarks to the Author): 

Deniz et al have mined publicly available data from AMLs, leukemic cell lines and 
monocytes/macrophages and investigated the frequency and identity of transposable elements at 
sites of chromatin accessibility, epigenetic marks of active/inactive promoters/enhancers to focus 
on a limited number of families to investigate their gene regulatory potential. As acknowledge, the 
concept is not novel, but this is an excellent example of using public data to explore a hypothesis 
and performing a series of valid experiments to elegantly prosecute their case. My comments are 
as follows. 
Figure 1D- were the expression levels averaged across BluePRINT AMLs? This wasn’t clear to me. 
Figure 2C- the presence/absence of a particular histone mark at a particular site was called using 
MACS2 with set thresholds. How were these normalised across samples? 
Figure 2F- Indicate more clearly in the figure that the profiles are of H3K27Ac and given the 
occasionally noisy profiles- mark the peaks that were called by MACS 
Figure 3E- Given the visual association that motifs were enriched at DHS elements, which the 
authors discount as insignificant- this should be backed up by valid statistics. There is publicly 
available Hi-C data in K562 cells. It should be possible to directly link the TF bound TEs with gene 
promoters. 
Figure 4B- Following CRISPR of elements in k562, gene expression values were shown for three 
ZNF genes. The displayed window in 4A only shows one presumably for practical considerations 
but it leaves the reader wondering whether RPL7L1 and BIK exist in gene deserts or selective data 
representation. Use of K562 Hi-C data could potentially show the TE/promoter links. Show 4B as in 
5G so the number of clones analysed is evident. 
Figure 5- deposition of dCas9 at intronic regions can disrupt transcript elongation independently of 
disrupting a regulatory element. What proportion of genes with intronic dCas9 (5B) showed 
reduced expression and could this have impacted on cell growth? 
Figure 6- I would be interested to know what the chromatic accessibility/histone profiles at LTR2 
adjacent to APOC1 look like in CD34+? 
General- 
There were a number of references in text that have not transferred to the reference list. This 
should be corrected. 
TF binding data is limited to K562 cells. There is publicly available TF data in CD34+ (BloodChIP) 
Have the authors looked for the presence of TEs at heptad binding sites? 
When focussing on one or other family of LTR from one figure to the next, the authors should 
justify the selection 
Legend 1- line 714- incomplete credit. Line 726- add colours against expression (orange), DHS 
peak (blue)