On the role of H3.3 in retroviral silencing

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Figure 1: Comparative analysis of ERV expression and histone modifications at ERVs in knockout ES cell lines.
Figure 2: PCR analysis of polymorphic IAP insertions.

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Gene Expression Omnibus


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G.W., T.S.M., M.C.L., D.L.M., K.O. and G.J.F. planned and designed the study. G.W., R.R., W.W., B.W., M.B. and R.K. performed experiments. G.W., M.M.K. and A.D.E. analysed the data. G.W. and T.S.M. wrote the manuscript with contributions from M.C.L., D.L.M. and G.J.F.

Corresponding author

Correspondence to Todd S. Macfarlan.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 H3.3 is incorporated into ERVs by the chaperone DAXX.

a, Top, representation of the two conditional Daxx-knockout alleles generated by CRISPR–Cas9-mediated loxP insertion at the indicated introns. Bottom, quantitative RT–PCR analysis of Daxx mRNA expression in Daxx-knockout ES cells. Data are mean ± s.d. expression (normalized to Gapdh) relative to the corresponding wild-type ES cells (n = 3, technical replicates). b, H3.3–YFP enrichment at 717 full-length IAP ERVs in two independently derived conditional Daxx-knockout ES cell lines.

Extended Data Figure 2 ERV deregulation in H3.3-knockout ES cells.

a, Mean fold change in ERV (LTR elements annotated in UCSC RepeatMasker) expression in H3.3-knockout ES cells (two cell lines) over wild-type ES cells (one cell line). b, Fold change in ERV expression comparing two H3.3-knockout ES cell lines. ERVs belonging to the IAP family are marked in red. Only ERV groups with more than 100 family members were considered for analysis.

Extended Data Figure 3 ERV expression in Setdb1-, H3.3- and Daxx-knockout ES cells.

The fold change in expression over the corresponding wild-type control is shown for the top 20 upregulated ERV annotations in Setdb1-, H3.3- and Daxx-knockout ES cells. Annotations including -int represent the internal regions, which are transcribed from the cognate 5′ LTR, of the annotated ERV. For example, ERVK10C-int is the internal region of ERVK10C elements, with flanking LTRs: RLTR10A, RLTR10B and RLTR10C (depending on the specific genomic copy), which are also presented among the graphs. Similarly, IAPEz-int is the internal region of IAPEz elements with flanking cognate LTRs: IAPLTR1_Mm and IAPLTR1a_Mm, which are also represented. As the internal region is much longer and transcribed across its length, this is the most useful annotation to consider for expression analysis. The following published RNA-seq GEO data were re-analysed: GSM727424 (Setdb1-knockout ES cells); GSM1428580 and GSM1428581 (H3.3-knockout ES cells). Only ERV groups with more than 100 family members were considered for analysis.

Supplementary information

Supplementary Methods

This file contains supplementary methods regarding Cell lines, Native ChIP-seq, RNA-seq, Re-analysis of RNA-seq and ChIP-seq data, PCR screening of polymorphic IAP insertions and Polymorphic IAP insertion genotyping. (PDF 112 kb)

Supplementary Table 1

Re-analysis of reported de novo IAP integrations in H3.3 KO ES cells. (XLSX 23 kb)

Supplementary Table 2

List of IAP integrations incorrectly annotated as ‘H3.3 KO only’ by Elsasser et al. (XLSX 9 kb)

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Wolf, G., Rebollo, R., Karimi, M. et al. On the role of H3.3 in retroviral silencing. Nature 548, E1–E3 (2017). https://doi.org/10.1038/nature23277

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