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closeReferee Comments: Referee #1
Posted by PLOS_ONE_Group on 30 Jul 2007 at 01:59 GMT
In this work, Miles et al. showed that intergenic transcription in the human β-globin locus occurs over a large region including several genes in the ORG cluster. RNA FISH analysis of primary cultured human erythroid cells showed that transcription throughout the ORG region occurs prevalently during S-phase, whereas transcription in the LCR and δβ sub-domains occurs in non-S phase cells. These subdomains are developmental regulated and enriched with active histone H3 modifications. These results link levels of non-S phase intergenic transcription with domain-wide active histone modifications to histone H3.
Previous work from the same group showed that the HBB locus contains chromatin subdomais developmentally regulated in terms of transcription and DNase I sensitivity and that transcription is cell cycle dependent (Gribnau et al., 2000). The results presented in this work are mainly based on these previous observations. However, the data here shown, although performed at higher resolution, validate previously published data (i.e. RT-PCR and RNA FISH) but they the lack of novelty. The finding that these subdomains change their levels of active histone marks during development is consistent with previous data (transcription and DNase I sensitivity). However, this is an expected result and not enough for the conclusions suggested by the authors: "these results link high levels of non-S phase intergenic transcription with chromatin domains that are highly enriched in active histone modifications". Moreover, cell cycle dependent transcription of these subdomains was previously shown (Gribnau et al., 2000) as well as intergenic transcription in the ORG cluster in the vicinity of the -236 LTR (Xiang et al. 2006). . The authors clearly demonstrated that transcription from the ORG cluster occurs during S-phase while LCR and δβ subdomains are transcribed predominantly in G1 phase independently of transcription in the ORG region and εγ domains. However, the conclusion that these results "link levels of non-S phase intergenic transcription with domain-wide active histone modifications to histone H3" is quite ambitious without having performed ChIP experiments on sorted and/or synchronized cells.
N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.
RE: Referee Comments: Referee #1
PFraser replied to PLOS_ONE_Group on 16 Aug 2007 at 11:34 GMT
The Reviewer is correct that the data presented here is at higher resolution and validates our previously published RNA FISH data. However we have not published developmental RT-PCR data on intergenic transcripts previously. These data were included in this manuscript mainly to validate our RNA FISH data, and thus confirm that the levels of RNA transcripts produced from the various sub-domains of the globin locus match the RNA FISH data on the differences in percentage of loci which show an intergenic signal. However we also wanted to respond to contradictory reports from another group, which suggested that intergenic transcription was fairly constant and did not vary across the locus based on nuclear run-on assays and their own RT-PCR data, which was for some unknown reason, presented on a log scale, which of course completely hides differences of 2- or 3-fold which we originally observed and felt important to show. So lack of novelty…maybe...but this data is unpublished and essential to the field.
Regarding the ChIP data, one could say (with hindsight) that it was expected that the histone modification domains would match the domains of DNase I sensitivity and high levels of intergenic transcription. Nevertheless these experiments had to be done and the results had to be shown; science is based on data and not on expectation.
We previously showed that intergenic transcription was cell cycle specific in the globin subdomains in Gribnau et al., 2000. It was shown to occur in early S phase but predominantly in G1 phase of the cell cycle. It is true that intergenic transcription has been shown to occur in the ORG cluster by others as we clearly state and reference in our manuscript. However what has not been shown, and is novel, is that the ORG cluster intergenic transcription occurs primarily in S phase while the active domain intergenic transcription occurs primarily in G1 phase. This is an important and novel finding which we discuss in relation to other current findings.
Finally, regarding our conclusions. We disagree with the Reviewer. We have shown quite clearly that there are domains of modified chromatin containing 'active' marks and that these correlate with domains of high-level, G1 phase specific intergenic transcription. This conclusion is sound and fully supported by our results. Performing ChIP on sorted or synchronized cells would not change that conclusion. What those experiment could help to elucidate is whether the S phase specific transcription which is rarer and appears to occur at a lower level or rate, results in transient or more rapidly turned over modification of the chromatin in the silent Org cluster. An interesting question, but not one that we have addressed in this paper.