Peer review of: A rare variant of African ancestry activates 8q24 lncRNA hub by modulating cancer (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
This study explores the potential role of a rare SNP that is seen at highest frequency in African 
populations and associated with a twofold prostate cancer risk as comparted to non-SNP carriers. The 
authors posit that there is a functional role that enables the initiation or development of prostate 
cancer through alterations in non-coding regions of the genome. Using 4C capture approaches to 
explore to DNA interactions they nominate as set of lncRNAs that are activated and may enhance the 
activation of MYC, a commonly activated oncogene in all prostate cancer. Validation was performed on 
the TCGA cohort. 
 
1) The authors are not entirely clear as to what prostate cancer Hi-C data was used for this study. To 
date there are few published cancer datasets. It would be important to specify the data set(s). There 
are also data sets using benign prostate cells (RWPE) with and without ERG expression, another 
important prostate cancer oncogene. Were these data also explored? Addition of these data would be 
useful to understand how universal these findings are in prostate disease progression. This is 
particularly of interest because SPDEF is also an ETS transcription factor that was identified. 
 
2) Exploration of the TCGA public data is important to attempt to validate their findings. Despite the 
small numbers of cases, they do see higher MYC levels in cases with the risk alleles. It would be 
important to clarify if these cases have or do not have MYC amplifications? This is a common region of 
genomic amplification in prostate cancer. 
 
3) It is not clear to this reviewer if the outcomes data are valid. The TCGA did not consistently collect 
outcome data. It this correct? What are the demographics of these cases used? 
 
4) The authors might extend these validation findings to some of the larger datasets that were 
generated on advanced prostate cancer (e.g., SU2C-PCF, WES, RNAseq data) or ICGC whole genome 
data in order to compare the risk allele or other stated associations. 
 
 
 
Minor Comments 
 
1) The abbreviation for DNase I hypersensitive sites, DHS, should be defined in the main text. Perhaps 
even briefly defined for the non-expert. 
 
2) The HiC data is an important inclusion in this study. The authors cite the Wang paper as the main 
reference, which is providing a convenient browser but is not the original description of Hi-C data. 
 
3) “ESMA experiments”. When possible please introduce acronyms to the non-specialist audience. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
This work by Walavalkar et al. describes the regulatory expression of a lncRNA hub modulating a 
prostate cancer associated enhancer. The authors asked the mechanistic function of a rare snp found 
in the 8q24 locus associated with greater susceptibility in prostate cancer. Using several ChIP-, 
ATTAC- and RNA-seq dataset published by different consortiums, they established that the rare 
variant is located within a region supporting enhancer features. Following cloning the different 
“natural” variants of this putative enhancer in front of a reporter gene, they show that it embeds 
transcription activation capacity. The analysis of HiC dataset and 4C single genes experiments indicate 
that the enhancer region is in close contact with lncRNA promoters associated with cMyc expression. 

TCGA clinical and expression dataset suggest that the enhancer variants are associated with poor 
outcome. Interestingly, the variants define a consensus binding site for the SPDEF androgen 
transcription factor. Chip experiments show that SPDEF can bind to these rare variants in vivo and 
support a model in which it further activates the lncRNA expression. 
Although the conclusions of the manuscript are very attractive, several major issues need to be 
addressed for publication in Nature communication, especially much more has to be presented 
concerning the clinical aspects and enhancer activities. 
 
Major issues/suggestions 
1. The luciferase test is poorly explained and has major variabilities that are not explained by the 
authors. For instance, the comparison between allele G and T without androgen show almost a 2 fold 
increase in Figure 2B, but none in Figure 2D (green points) and 4 fold change in Figure 5D. These 
variations strongly compromise the reliability of such measurements. A lentivirus integration might 
help to get more robust results. In figure 2, it should be presented the vector and the internal 
normalization process, since it is very complicated to understand for the reader. As it is, it shadows 
the demonstration the the alleles have a significant differences for the enhancer activity- a key 
message of the paper. What about chromatin data to support further opening of the region (attac, 
Mnase etc..) in the different allele conditions? 
2. “eRNA expression from the enhancer is not changed”. This quite strange since eRNA are supposed 
to be cryptic RNA. Their role as enhancer is more about the chromatin state and TF binding capacity, 
rather than the eRNA expression itself. In addition, the eRNA POLII might be activated but the eRNA 
rapidly degraded. So It would not be a surprise that whatever the enhancer is active or not, the eRNA 
(already very low) levels remain constant. 
3. The comparison with breast tumor TAD (sup S2A) should be properly presented and the figure 
suppS2A placed in the main part. 
4. Figure 4D and 4E are completely unbalanced and cannot be used for the model since the statistics 
are nowhere near any relevance! 737 cases compared with 15 and 1! The authors should find a beeter 
cohort or engage their own cohort if they want to draw any clinical conclusion. I suspect that the 
absence of the “n” number for Figure 4F would reflect also very poor numbers. If this is the case, it 
should be removed or placed in supplementary file to be only discussed as preliminary insights. 
Without good clinical data, the manuscript would miss an important message and could not be 
accepted in Nature communication. 
 
5 Figure 5B missed 2 important controls: with a scramble oligo and using cellular extract of cells 
treated with siSPDEF. 
6. The experiment in Figure 5C is also a crucial aspect of the manuscript and as it is, it fails to 
convince the reader if SPDEF binds differently the different alleles. The ChIP analysis has no negative 
or positive controls to determine whether the SPDEF signals are significant or not. The author should 
perform the experiment by probing SPDEF known promoters and other negative regions of the 
genome. Additionally, a normalization gene should be used to confirm that the IP is robust. 
7. the competition experiment is very interesting but requires a more drastic dose response. The 
authors should perform a proper competition by increasing different doses (and measuring it in vivo) 
of the competitive plasmid/allele, and not just one. 
Minor comments/questions 
1. figure 1D some tumors have no signal for ATAC seq, is there any reason? Gleasons? Clinical 
specificity of these tumors? 
2. typo line 183 “loos” instead of loops 
3. typo line 225 “(Fig. (Fig 4C)” 
4. Figure 2C, the bottom of the figure with chip data of FOXA1, DHS or K27ac are not discussed at all. 
remove 
5. In figure 5D, 3Xflag SPDEF should be at the top of the gel. 
6. Figure 5G. the legend is missing: is it fold change luciferase? And on the X axis, is it ratio of the 
plasmids? It should be indicated and the results better described in the text.