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Referee comments: Referee 1

Posted by PLOS_ONE_Group on 07 Mar 2008 at 18:42 GMT

Referee 1's review:

The authors study DNA methylation and expression of avian leukosis virus subgroup E (ALVE) variants and one ALV subgroup B variant (TVB) in two different chicken strains, one resistant and one susceptible to virus-induced tumorigenesis. They find convincing strain- and tissue-specific differences in CpG methylation at the loci. Further, they demonstrate the differences in CpG methylation are in many cases inversely correlated with expression at the loci. The methods appear sound, and both the number of animals studied and statistical analyses seem reasonable.

My major concerns are regarding the authors' description and interpretation of their results. Regarding the correlations between methylation and expression, the authors repeatedly claim evidence that methylation is inducing transcriptional repression. This is not substantiated by the data; references to causality should be omitted. Also, the authors claim to have evidence of 'epigenetic inheritance' based upon studies of epigenetic differences at the loci among backcrossed animals generated from the inbred lines. A more likely explanation is that the epigenetic differences are secondary to genetic variation generated by the backcrossing strategy. References to epigenetic inheritance should be omitted.

It is unclear how many copies of the ALVE and TVP loci are represented in each of the chicken genomes studied, and whether these numbers may differ substantially among the strains. It may help to comment on this and provide any available data.

Another concern is that it is not clear the authors are distinguishing between CpG sites and CpG islands. For example, in the abstract they refer to studying "3-6 CpG islands" of the four conserved regions. In fact, they quantified methylation at 3-6 CpG sites. If the authors are intentionally referring to CpG islands (as opposed to CpG sites), they should make it clear. For example, the locations of CpG islands could be indicated in Fig. 1, if intended.

The discussion is too long. The authors should succinctly focus on clarifying the overall interpretation and significance of their data and suggest unresolved issues for future study.

The quality of some of the figures is not very high. For example, the labeling and numbering of the axes in Fig. 10 are cryptic and inconsistent with the journal format. Also, the manuscript would benefit from editing for English grammar.

N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.