The long non-coding RNA Dali is an epigenetic regulator of neural differentiation

Thank you for sending your work entitled "The DNMT1 associated lncRNA Dali is an epigenetic regulator of neural differentiation" for consideration at eLife. Your article has been favorably evaluated by Detlef Weigel (Senior editor) and 2 reviewers, one of whom is a member of our Board of Reviewing Editors. The Reviewing editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

Your work focuses on the functional roles of the lncRNA Dali and its regulation not only of its neighboring transcription factor gene Pou3f3, but importantly also at distal sites through the methylation of these sites. The topic of the functional roles of lncRNAs is of keen interest and most importantly the mechanism of how these roles are achieved is of critical importance. Thus, this manuscript is both timely and important. However, there are several issues that the authors should make clearer for the readers in order for an audience to understand the messages being communicated and to provide a clear and supportable set of conclusions. These issues are:

1) Several places in the manuscript the authors' interpretation of their data results in a set of specific conclusions about mechanisms or cause and effect relationships that are not the only explanation or conclusion. Importantly, there is no acknowledgement of other interpretations or explanations of why other interpretations cannot be concluded. For example, while the suggestion of recruitment of Dali via a complex of DNA binding proteins that include CTCF is an interesting hypothesis, this collection of data can be interpreted by alternative mechanisms such as the coincident binding of CTCF (due to CTCF genome-wide and abundant binding).

2) What was the basis of choosing 10 Dali binding sites? What changes, if any occur, at the other 5 sites? Given that half of the results obtained are consistent with the model of Dali or Dali:Pou3f3 complex acting in trans to reduce DNMT1 methylation, there appears to be the likelihood that the mechanism may minimally involve a more complex set of interactions. This should be acknowledged.

3) “…we performed Combined Bisulfite Restriction Analysis (COBRA) (Xiong and Laird, 1997) in parallel at 10 different CpG islands and demonstrated that five of these regions (corresponding to 4 genes) exhibited altered restriction profiles indicative of altered DNA methylation status after Dali depletion […] These results are consistent with Dali (or a Dali:POU3F3 complex, see below) acting in trans to reduce DNMT1-mediated CpG methylation at a subset of bound and regulated gene promoters away from its site of transcription”: this is speculation since the effect on the use of the 5' most CpG site could be achieved through a secondary effect of the Dali KD.

4) Figure 2E (heat map); are these the results of all 3 KDs or only1? Given the results seen in Figure 2E what data indicates that some or most of these expression changes are not explained by "off-target" effects by the sequences used in the KDs?

1) Several places in the manuscript the authors' interpretation of their data results in a set of specific conclusions about mechanisms or cause and effect relationships that are not the only explanation or conclusion. Importantly, there is no acknowledgement of other interpretations or explanations of why other interpretations cannot be concluded. For example, while the suggestion of recruitment of Dali via a complex of DNA binding proteins that include CTCF is an interesting hypothesis, this collection of data can be interpreted by alternative mechanisms such as the coincident binding of CTCF (due to CTCF genome-wide and abundant binding).

We have now edited the main text to include additional explanations that are also consistent with the data as suggested by the reviewers. This specific example now states that Dali and CTCF “might independently bind adjacent sequence, or compete for binding to the same region”.

2) What was the basis of choosing 10 Dali binding sites? What changes, if any occur, at the other 5 sites? Given that half of the results obtained are consistent with the model of Dali or Dali:Pou3f3 complex acting in trans to reduce DNMT1 methylation, there appears to be the likelihood that the mechanism may minimally involve a more complex set of interactions. This should be acknowledged.

We selected these 10 Dali binding sites to maximise the likelihood of detecting DNA methylation changes using COBRA. We ensured that each region contained several COBRA-compatible restriction enzyme sites and could be efficiently amplified from bisulfite-converted template.

We also now indicate that the failure to detect changes at all regions tested could reflect either that the DNA methylation status of the remaining regions did not change upon Dali depletion or that those changes that occurred were undetected due to technical limitations of the COBRA assay.

We agree with the reviewers that Dali mediated DNA methylation changes in trans may minimally involve a more complex set of interactions that are in addition to Dali, POU3F3 and DNMT1. This is acknowledged in the revised manuscript as suggested. We now state: “Although other unidentified factors are also likely to play a role, our results are consistent with Dali (or a Dali:POU3F3 complex) acting in trans, as part of a multi-subunit ribonucleoprotein complex, to reduce DNMT1-mediated CpG methylation at a subset of bound and regulated gene promoters away from its site of transcription.”

3) “…we performed Combined Bisulfite Restriction Analysis (COBRA) (Xiong and Laird, 1997) in parallel at 10 different CpG islands and demonstrated that five of these regions (corresponding to 4 genes) exhibited altered restriction profiles indicative of altered DNA methylation status after Dali depletion […] These results are consistent with Dali (or a Dali:POU3F3 complex, see below) acting in trans to reduce DNMT1-mediated CpG methylation at a subset of bound and regulated gene promoters away from its site of transcription”: this is speculation since the effect on the use of the 5' most CpG site could be achieved through a secondary effect of the Dali KD.

Thank you. We now have added: “The preferential use of the 5́’ most CpG site could reflect a secondary effect of Dali knockdown. Nevertheless, the observation that this site is bound by Dali transcript suggests that Dali may function by promoting the preferential use of a distantly located (and more rarely used) alternative promoter potentially through its effect on promoter-associated CpG island methylation.”

4) Figure 2E (heat map); are these the results of all 3 KDs or only1? Given the results seen in Figure 2E what data indicates that some or most of these expression changes are not explained by "off-target" effects by the sequences used in the KDs?

The data in Figure 2E were generated from three independent Dali knockdowns. To assess whether these expression changes are likely to be explained by ‘off-target’ effects we used two further shRNA expression constructs targeting different regions of the Dali transcript to deplete Dali expression and validated expression changes in 14 out of 15 Dali targets identified in the microarray. These results are shown in Figure 2-figure supplement 1 panel C and in the modified text.