We would like to make an important correction to the paper entitled “Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells”, Nature, 2015. It is available at Nature here (download PDF).
In the 2015 study, we provided biochemical and genetic evidence that the histone variant H3.3 plays a role in the maintenance of interstitial heterochromatin and transcriptional silencing at specific classes of endogenous retroviral elements (ERVs) in mouse embryonic stem cells (ESCs). In the last experiment (Figure 4d), we reported evidence for retrotransposition of IAP elements in the absence of H3.3, using a de novo mapping approach from paired-end sequencing data comparing a wildtype and knockout cell line. We identified elements that were not annotated in the mm9 reference genome (C57Bl/6), and either in H3.3 KO, WT, or both cell lines. After publication of our manuscript, it was brought to our attention that the majority of elements in either of those categories overlaps with known IAP integration sites in non-C57Bl/6 strains. The H3.3 KO ESCs used in our original study were generated from a mixed background (129xC57Bl/6) ESC line.
Matching all sites called de novo by our analysis against published2,3 and unpublished reference sets, we indeed identified 97 of 116 to be present in at least one reference mouse strain, predominantly 129. We therefore conclude that these sites are preexisting in our mixed ESC background and did not arise as a result of H3.3 knockout.
Of the remaining, 17 putative insertions were called unique to H3.3 KO1. To test if these sites arose as result of H3.3 knockout, we PCR genotyped the elements in the WT control and both H3.3 KO1 and H3.3 KO2 cell lines. The two KO cell lines were derived independently from an h3f3aFl/Fl intermediate. The intermediate has reduced levels of H3.3.Any integrations arising in H3.3 KO1 after complete H3.3 deletion should not be present in KO2. However, we observe a nearly complete overlap between H3.3 KO1 and H3.3 KO2 genotyping results (Figure 1 of our reply).
Thus, none of the putative novel insertions can be attributed to a transposition event after complete H3.3 knockout.
We conclude that our method to de novo map IAP integration sites using paired-end sequencing of genomic DNA does not provide evidence for increased IAP mobility in H3.3 knockout cells. We note that, to be detectable by our method, an insertion site has to be present in a considerable fraction of the cells in the population. This technical limitation highly favors identification of preexisting sites in populations derived from a single clone over subsequent spontaneous and stochastic transposition events. We apologize for our mistake interpreting the data presented in Figure 4d and we would like to correct the conclusions drawn regarding IAP mobility in our original manuscript, namely in the penultimate sentences of the abstract, and penultimate paragraph of the main text. All other conclusions towards an important function for H3.3 within interspersed heterochromatin, namely the marked reduction of the silencing-associated modification H3K9me3 and KAP1 occupancy in H3.3 knockouts, together with an upregulation of specific ERV transcripts, remain unaffected.
In the process of validating all major results, we have integrated existing and new RNA-Seq datasets, confirming our original model. We observe modest but consistent desilencing of specific ERV transcripts including IAP after depletion or in the absence of H3.3, DAXX, and ATRX.
Direct links to relevant datasets:
RNA-Seq ATRX and DAXX KO cells
GSE73881 Transcriptome analysis of ATRX and DAXX knockout mouse ESC (2015)
RNA-Seq DAXX KO cells, from other groups
GSE70965 ERV reactivation in Daxx knock-out embryonic stem cells (2015)
GSE70849 The DAXX/ATRX Complex Protects Tandem Repetitive Elements in the Absence of DNA Methylation by Promoting H3K9 Trimethylation (2015)
- Elsässer, S. J., Noh, K.-M., Diaz, N., Allis, C. D. & Banaszynski, L. A. Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells. Nature 522, 240–244 (2015).
- Nellåker, C. et al. The genomic landscape shaped by selection on transposable elements across 18 mouse strains. Genome Biol. 13, R45 (2012).
- Zhang, Y., Maksakova, I. A., Gagnier, L., van de Lagemaat, L. N. & Mager, D. L. Genome-wide assessments reveal extremely high levels of polymorphism of two active families of mouse endogenous retroviral elements. PLoS Genet 4, e1000007 (2008).