Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun;27(6):913-921.
doi: 10.1101/gr.215830.116. Epub 2017 Mar 24.

PML protein organizes heterochromatin domains where it regulates histone H3.3 deposition by ATRX/DAXX

Affiliations

PML protein organizes heterochromatin domains where it regulates histone H3.3 deposition by ATRX/DAXX

Erwan Delbarre et al. Genome Res. 2017 Jun.

Abstract

Maintenance of chromatin homeostasis involves proper delivery of histone variants to the genome. The interplay between different chaperones regulating the supply of histone variants to distinct chromatin domains remains largely undeciphered. We report a role of promyelocytic leukemia (PML) protein in the routing of histone variant H3.3 to chromatin and in the organization of megabase-size heterochromatic PML-associated domains that we call PADs. Loss of PML alters the heterochromatic state of PADs by shifting the histone H3 methylation balance from K9me3 to K27me3. Loss of PML impairs deposition of H3.3 by ATRX and DAXX in PADs but preserves the H3.3 loading function of HIRA in these regions. Our results unveil an unappreciated role of PML in the large-scale organization of chromatin and demonstrate a PML-dependent role of ATRX/DAXX in the deposition of H3.3 in PADs. Our data suggest that H3.3 loading by HIRA and ATRX-dependent H3K27 trimethylation constitute mechanisms ensuring maintenance of heterochromatin when the integrity of these domains is compromised.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Loss of PML partitions histone H3.3 into a compact micrococcal nuclease (MNase)–resistant chromatin compartment. (A) MEF fractionation into a cytosolic fraction (S1), a Triton X-100 (0.5%)-insoluble/MNase (0.5 U/µg DNA)-soluble fraction (S2), and a Triton X-100/MNase-insoluble fraction (P2). (B) Nucleosome laddering (DNA; top) and Western blot (bottom) of endogenous H3.3 and total H3 in each fraction, from wt and Pml ko MEFs. (C) Proportions of H3.3 and total H3 in P2; (*) P < 0.05 relative to wt; paired t-tests. (D) Distribution of H3.3 and PML in S1, S2, and P2 from mock-transfected wt and Pml ko MEFs and from ko MEFs expressing MYC-PML1 or MYC-PML2 (full-length PML); alpha-tubulin was used as loading control for S1. (E) Proportions of H3.3 and total H3 in P2 after knockdown of Hira, Daxx, or Atrx; mean ± SD, three experiments; (*) P = 0.03 (siHira wt); 0.04 (siAtrx wt); 1.4 × 10−5 (siHira ko); (§) P = 0.06 (siDaxx, wt); paired t-tests relative to mock control.
Figure 2.
Figure 2.
Loss of PML accelerates deposition of epitope-tagged H3.3 into chromatin. (A) Distribution of H3.3-mCherry (mC) in wt and Pml ko MEFs 24 h after H3.3-mC transfection. Cells were labeled using anti-PML antibodies and DNA stained with DAPI. Bar, 10 µm. (B) Proportions of cells of “type 1” and “type 2” over time after H3.3-mC transfection; no “type 2” Pml ko cells are detected at any time point. (C) Expression of HA-PML (full-length isoform PML2) in Pml ko MEFs restores H3.3-mC targeting to PML bodies. Bar, 10 µm. (D) FRAP analysis of H3.3-EGFP in wt and Pml ko cells; mean ± SD for 19–33 cells of each indicated type. See Supplemental Figure 2C for visualization of photobleached areas. (E) Western blot of H3.3-Flag and endogenous H3 in a 1% Triton X-100 soluble (Sup.) and insoluble (Pellet) fraction in wt and Pml ko MEFs.
Figure 3.
Figure 3.
PML associates with large heterochromatin domains. (A) ChIP-seq profiles of PML in wt and Pml ko MEFs and of H3K9me3, H3K27me3, and H3.3-Flag in wt MEFs; data are shown as log(ChIP/input) ratios. RNA-seq counts (FPKM) are shown. (B) PAD length (median, 0.8 Mb). (C) Expression levels of genes enriched in H3.3-Flag, H3K27me3, and H3K9me3; genes in PADs; and of all RefSeq genes in wt MEFs. (D) Gene density within H3.3-Flag peaks, in PADs, and in the whole genome. (E) Immuno-FISH analysis of PML (showing PML bodies; immunolabeling, red signal) and PADs (green signal; arrows; FISH probes). Ube2b and Gapdh are shown as genes outside PADs. Planar and orthogonal views are shown. Bar, 5 µm. Graph indicates distribution of FISH probe distances to nearest PML body (about 60 FISH signals analyzed). (F) ChIP-qPCR of H3.3-Flag and total H3 in indicated PAD and non-PAD regions in wt MEFs. (*) P < 0.01 relative to H3.3-Flag at all other sites; two-tailed t-tests; mean ± SD, three experiments. See Supplemental Table 4 for position of amplicons. (G) PAD area coverage by H3K9me3 and/or H3K27me3 peaks in wt cells.
Figure 4.
Figure 4.
The heterochromatic state of PADs is remodeled in the absence of PML. (A) Profiles of H3K9me3, H3K27me3, and H3.3-Flag inside and outside PADs in wt and Pml ko MEFs. (B) Median enrichment level of indicated marks in PADs and in randomized PADs. (*) P < 2.2 × 10−16; Wilcoxon tests. (C) Proportions of PAD area enriched in H3K9me3 and H3K27me3. (D) Fold change of H3K9me3 and H3K27me3 coverage in PADs (red bars), between PADs (purple bars), and in the whole genome (brown bars) in Pml ko versus wt cells. Reference (wt) coverage is set to one (blue bars). (*) P < 0.01, (**) P < 0.001 relative to wt; one-sample t-tests.
Figure 5.
Figure 5.
Absence of PML promotes H3.3 loading by HIRA in PADs. (A) ChIP-qPCR of H3.3-Flag in PAD and non-PAD sites in wt and Pml ko MEFs after Hira, Daxx, or Atrx knock-down. (B) ChIP-qPCR of H3K27me3 under the same conditions as in A. All ChIPs: mean ± SD of three to five experiments. (*) P < 0.02; Fisher's exact tests. Position of amplicons is given in Supplemental Table 4. (C) PML organizes heterochromatin domains and modulates H3.3 loading. (Left) In wt cells, PML associates with heterochromatic H3K9me3-rich domains (PADs). Deposition of H3.3 in PADs is low relative to inter-PAD regions, and can be mediated by HIRA, DAXX, or ATRX. (Right) In the absence of PML, H3K9me3 levels are reduced in PADs. The ATRX/DAXX complex loses is ability to load H3.3 in these regions. In contrast, HIRA retains its ability to deposit H3.3 in PADs, supporting the idea of a gap-filling mechanism (Ray-Gallet et al. 2011) established to preserve chromatin integrity. A heterochromatic state is maintained in PADs by an increase in H3K27me3 in a manner implicating the ATRX/DAXX complex, perhaps through the ability of ATRX to recruit the PRC2 complex to chromatin (Sarma et al. 2014). Our results suggest an interplay between the HIRA-H3.3 and ATRX/DAXX-Polycomb pathways to maintain heterochromatin homeostasis in domains where H3K9me3 is compromised in the absence of PML, with the purpose of keeping these regions transcriptionally silent.

Similar articles

Cited by

References

    1. Adam S, Polo SE, Almouzni G. 2013. Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA. Cell 155: 94–106. - PubMed
    1. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25–29. - PMC - PubMed
    1. Banaszynski LA, Wen D, Dewell S, Whitcomb SJ, Lin M, Diaz N, Elsasser SJ, Chapgier A, Goldberg AD, Canaani E, et al. 2013. Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells. Cell 155: 107–120. - PMC - PubMed
    1. Bernardi R, Pandolfi PP. 2007. Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat Rev Mol Cell Biol 8: 1006–1016. - PubMed
    1. Chang FT, McGhie JD, Chan FL, Tang MC, Anderson MA, Mann JR, Andy Choo KH, Wong LH. 2013. PML bodies provide an important platform for the maintenance of telomeric chromatin integrity in embryonic stem cells. Nucleic Acids Res 41: 4447–4458. - PMC - PubMed

MeSH terms