Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L‐dose dependency in HDAC1‐deficient thymic lymphoma

Abstract DOT1L methylates histone H3K79 and is aberrantly regulated in MLL‐rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1 Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.

Thank you for submitting your manuscript for consideration by The EMBO Journal and I apologize again for the delay of the reviewing process. We had been waiting for the third referee report to come in, but have now decided to move ahead with the two referee reports on your study, which are included below.
As you will see, both reviewers expressed an overall interest in the study, but referee #2 does raise some major concerns that would have to be fully addressed in a revised version of the manuscript. S/he in particular points out that the contribution of Rpd3's deacetylase activity should be assessed in more detail, as well as its role as a transcriptional repressor.
Should you be able to fully address these specific concerns, as well as the various additional issues raised, then we would be happy to consider this study further for publication. I would therefore like to invite you to prepare and submit a revised manuscript. This study identifies a link between RPD3 and Dot1 using very sophisticated screening strategy in yeast that had previously been developed by the same group. In depth description of genetic mutants confirms convincingly this prediction that the Rpd3L complex negatively regulated H3K70 methylation. Importantly the authors show that the same dependency exists in the mouse and further test if this also plays a function in thymic lymphomas. This reveals that reducing the dose of Dot1 increases lymphoma in an HDAC dependent manner and that Hdac1 deficient thymic lymphomas lines require Dot1. This a very clearly written manuscript. Experiment and data appear to be of very high quality.
The study provides a novel chromatin link to regulation of H3K79 methylation that is conserved from yeast to man. One of its strength is the combination of clever functional screening in a model organism and readily translating this into the complexity of cancer, where evidence for chromatin modifiers is ample but little knowledge exists how these influence tumor formation. This creates important new opportunities to gain insights into this problem and a path to use sensitive and highly controlled experiments in a model organism can generate hypothesis that can be tested in a disease setting.
Referee #2: The manuscript by Vlaming, McLean et al. uncovers a functional relation between the histone deacetylase Rpd3 and methylation of histone H3 K79 by Dot1. They show that in S.cerevisiae a deletion of members of the Rpd3L complex results in increased H3K79me3 as well as H2B ubiquitination and transcription. But while H2B ubiquitination directly correlates with transcription, increased K79me3 better correlates with binding of Rpd3.They then go on to show that conditional deletion of HDAC1 in mouse thymocytes causes thymic lymphomas that depend on Dot1 dosage, as its presence prevents apoptosis.
Major concerns: -The correlation of increased H3K79me3 in the absence of rpd3 and Rpd3 binding data makes a very compelling point for a direct regulation of H3K79 methylation by Rpd3. What is not clear is to what extent its catalytic activity as an HDAC plays a role. Given the many examples of cross-talk between histone modifications, it would be important to understand if Rpd3's regulation of H3K79 methylation is mediated by histone acetylation. (The authors refer to the cross talk between H4K16 acetylation and H3K79 methylation in their discussion, but H4K16 is not a known substrate for Rpd3 in yeast.) It therefore would be of great interest to determine if H3K79me3 is equally increased in a catalytically dead rpd3 mutant.
-Deposition of H3K79me3 has been linked to active transcription. Given the function of Rpd3 as a transcriptional repressor the role of transcription in increased K79 methylation at the 5' end of genes should include pervasive transcripts. In particular antisense transcription, as there is a direct anticorrelation between the presence of such transcripts and H3K79me3  Figure 2 A: the lack of increased H2Bub and H4ac of group III genes (or at least part of them) could be a technical problem due to differential strength of antibodies. This possibility should be discussed in the text; Also, the units for the y-axis should be indicated in the figure. Also, it would be more appropriate to describe the ranking based on "gene expression" rather than "transcription", given that this does not account for all forms of transcription. Text: Page 7 line 159: "To test whether H3K79me-regulated genes were direct Rpd3 targets...." This is misleading, as it could be interpreted that the expression of these genes is regulated by H3K79 methylation.
Additional suggestions: Finally, the authors show that Hdac1-deleted thymic lymphomas are dependent on the dosage of Dot1. This raises the question whether increased H3K79 methylation is the major cause of these lymphomas or if other deacetylation driven events are necessary for tumor formation. It therefore may be interesting to determine if Dot1 overexpression mimics the effect of the deletion of HDAC1 on tumorigenesis. Rpd3. This analysis showed that INO80 does not specifically affect H3K79 methylation at genes that are also regulated by Rpd3. This finding is in agreement with the very large number of genes that is affected by INO80, whereas Rpd3 regulates H3K79 methylation at a subset of genes. We describe these analyses on pages 8-9 and Fig. EV2F.
Minor concerns: Figure 1 D and F: the y-axis title should be the same > The axes of these panels are indeed not the same; panel D is ranked on K79 me3/me1 in wild type while panel F is ranked on K79me3 in rpd3d/WT. We now emphasize in the text on page 7 that panel F represents a heatmap of H3K79me3 changes to avoid confusion.    > We thank the reviewer for this suggestion. We have made some attempts to overexpress DOT1L in mammalian cells and experienced problems in obtaining stable lines. Therefore, this approach will require more optimization. One major risk of this approach is that global overexpression of DOT1L may not mimic the increase in H3K79 methylation at specific target genes in HDAC1 mutants. Given these challenges and uncertainties as well as the substantial time investment, we believe that this interesting experiment falls beyond the scope of the current manuscript. 3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe.
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For the mouse experiment, a power analysis suggested group sizes of 20-25 mice. For other experiments, no explicit power analysis was used because effect size and standard deviation could not be estimated beforehand. Sample sizes were decided on case-by-case basis and are reported in the figure legends.
graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

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For the mouse experiment, a power analysis suggested group sizes of 20-25 mice. For other experiments, no explicit power analysis was used because effect size and standard deviation could not be estimated beforehand. Sample sizes were decided on case-by-case basis and are reported in the figure legends.
As described in the manuscsript. Mice that died without a lymphoma were censored in the survival curves. Mice of which the cause of death could not be determined were left out of the survival curve, together with their littermates. These criteria were previously established and based on previous studies, e.g. Heideman 2013. The health status of the animals was determined by animal caretakers, who were blind to the study design.
The health status of the animals was determined by animal caretakers, who were blind to the study design.

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