The human leukemia virus HTLV-1 alters the structure and transcription of host chromatin in cis

Reviewer #1:

[…] Overall, this is an interesting and well performed study. The manuscript could be improved by considering the following points:

1) The results clearly show that the CTCF binding site in the viral genome can participate in looping with cis host genomic elements, but whether this alters host genomic looping as the authors suggest is less clear. Can the authors define the loop patterns that would be present in the region of the integration site when no provirus is present?

This is indeed an interesting point: it is possible that HTLV-1 destroys normal chromatin loops as well as creating abnormal loops. However, the appropriate method to use to systematically map the loop patterns in the normal genome is HiC, which in principle identifies loops (albeit with limited sensitivity) between any two genomic regions (‘all to all’ mapping), whereas 4C identifies loops made between a defined ‘viewpoint’ and all other regions (‘one to all’ mapping). It is outside the scope of this study to carry out HiC analysis in addition to the 4C.

2) It would be helpful to provide a schematic of the authors' modifications to the 4C method.

Thank you for the suggestion. We have made a schematic diagram to compare conventional 4C and our modified version, q4C, and this is included as Supplementary file 2.

3) How have the quantitative aspects of this method been validated?

Quantification of chromatin looping. 4C (and indeed other ‘C techniques’) gives relative quantification of chromatin looping: in the absence of single-cell 4C – which no-one has yet developed – absolute quantification of loop frequency is not yet possible. Therefore, validation of the (relative) loop frequency depends primarily on the reproducibility of peak identification in independent biological replicate experiments, as we report in the present Results. The improvement in quantification introduced by the use of sonication instead of restriction enzyme digestion was demonstrated in our high-throughput analysis of retroviral integration sites (Blood 2011: 117, 3113; Bioinformatics 2012: 28, 755; et seq.)

4) The altered chromosomal looping and consequent effects on host gene transcription will be different in each clone with a different integration site. Overall, it is not clear how this affects HTLV-1 persistence or oncogenic potential. Some discussion of these points would strengthen the manuscript.

The impact of the provirus on chromatin looping and host transcription is indeed clone-specific. Because of the great clonal diversity of HTLV-1-infected cells in each host, identifying the impact of altered chromatin looping on HTLV-1 persistence or oncogenic potential will depend largely on bioinformatic analysis: in particular, to compare the association between CTCF binding sites and proviral integration sites between clones infected in vitro – in the absence of in vivo selection – and those that persist in vivo, and between malignant infected (ATL) clones and non-malignant clones. We are now embarking on this analysis. To emphasize the point raised by the reviewer, we have added the following sentence to the Discussion section:

“The altered chromosomal looping and consequent effects on host genome transcription depend on the proviral integration site, and so differ in each infected T-cell clone. Further work is needed to investigate the impact of altered looping and transcription on the persistence, infectivity and oncogenic potential of the infected T-cell clones.”

Reviewer #2:

[…] All in all, I found this to be a well-written and convincing study, certainly worthy of publication in eLife. My comments are mostly just points that might benefit from further discussion.

1) Although the primary observations seem quite compelling, the authors seem to tiptoe around the role of chromosomal looping in oncogene activation. There must be additional evidence in support of such a model that they can cite, such as the existence of oncogenic cell lines with aberrant expression of known oncogenes within a few Mbp of an HTLV-1 provirus. The focus of this study on non oncogenic lines makes it seem like they are splitting off an important part of the story.

Relationship of proviral integration to oncogene activation. This is of course a very important point. However, the chief aim of the present paper was to show that insertional mutagenesis – both local and at distant sites – is a general feature of HTLV-1 infection, and yet does not usually cause oncogenic transformation. We now plan to apply the techniques reported here to analyse chromatin looping and host transcription in cases of adult T-cell leukemia/lymphoma. In this planned study, we will focus on primary cells from ATL cases, rather than long-term in vitro cell lines, because ATL clones are notoriously difficult to propagate in vitro, so the relationship between such in vitro cell lines and primary ATL clones is at best uncertain. See also the response to reviewer 1, point 4.

2) It seems highly unlikely that the CTCF binding site is in the provirus by accident. Some discussion – even speculation – to this point would be very welcome.

We agree wholeheartedly with the reviewer’s implication that CTCF binding must confer some benefit on HTLV-1. However, this has proved a very difficult problem. To clarify the point, as the reviewer suggests, we have added the following short paragraph to the Discussion:

“We postulate that CTCF confers a survival or replication advantage to HTLV-1, because of the high degree of sequence conservation of the proviral CTCF binding site and the coincidence of the binding site with a border of epigenetic modifications associated with transcriptional activity [Satou et al., 2016]. However, the nature and mechanism of this putative advantage are not yet known.”