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Review
. 2020 Dec 2;48(21):11890-11912.
doi: 10.1093/nar/gkaa828.

PML nuclear bodies and chromatin dynamics: catch me if you can!

Armelle Corpet  1 , Constance Kleijwegt  1 , Simon Roubille  1 , Franceline Juillard  1 , Karine Jacquet  1 , Pascale Texier  1 , Patrick Lomonte  1
Affiliations
Review

PML nuclear bodies and chromatin dynamics: catch me if you can!

Armelle Corpet et al. Nucleic Acids Res. .

Abstract

Eukaryotic cells compartmentalize their internal milieu in order to achieve specific reactions in time and space. This organization in distinct compartments is essential to allow subcellular processing of regulatory signals and generate specific cellular responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells organize the distribution of macromolecules via various membrane-less nuclear organelles, which have gathered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less organelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this review, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the structure and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance.

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Figures

Figure 1.
Figure 1.
Structure of PML and organization of PML NBs. (A) Structure of the PML protein scaffold. All PML isoforms (I–VII), ranging from 882aa (PML-I) to 435aa (PML-VII), possess a conserved RBCC/TRIM motif in their N-terminal part. The different C-terminal parts of PML-I to VI are generated through alternative splicing of the 3′ exons 7 to 9 of the unique PML gene, while PML-VII only possesses exons (1–4 and 7b). SUMO modification sites (S) are indicated at lysine positions K65, K160, K490 and K616. The NLS (Nuclear Localisation Sequence) and the SIM (SUMO Interacting Motif) are indicated. NB: PML structure is not to scale. (B) Formation of canonical PML NBs. (1) Oxidative stress triggers PML cross-linking by disulfide bond formation. (2) Together with RBCC weak non-covalent interactions, this triggers oligomerization of non SUMOylated PML proteins. (3) UBC9-mediated (poly-)SUMOylation of PML then allows multiple SUMO-SIM interactions, (4) which stabilize the formation of the self-organized matrix-associated outer shell, possibly involving liquid-liquid phase separation mechanisms. Of note SUMO1 modification (yellow) is mostly present in the PML NB outer shell, while the poly-SUMO2/3 chains (orange) present in the shell also protrude to variable degrees in the interior of the PML NB. (5) Client proteins are recruited in the outer shell (eg Sp100 not shown) as well as in the inner core through specific interactions of their SIM with the SUMOylated-PML scaffold. (6) UBC9 SUMOylation of client proteins then enforces their sequestration in PML NBs. Turnover of client proteins is relatively rapid ranging from seconds to a few minutes. (C) PML NBs are interspersed in the chromatin. (left) Immunofluorescence analysis of human primary BJ fibroblasts stained by PML (green) and DAPI (red). Scale bar is 10μm. (right) Scheme showing PML NBs (green) surrounded by chromatin loops (red). Cellular loci, such as telomeres, can localize partly within PML NBs in specific cases (i) (see main text). Chromatin-related factors (histone modifiers, histone readers and histone chaperones) as well as viral genomes (ii) localize inside PML NBs.
Figure 2.
Figure 2.
Three possible functions of PML NBs in relation to their liquid-like properties. Liquid properties are advantageous for the cells by providing the ability of fast and easy rearrangements of macromolecules. Yet, the separation of the “liquid" nucleoplasm in several membrane-less condensates including PML NBs is essential to allow the formation of small reaction volumes with a different composition from the outside. Description of PML NBs as biomolecular condensates can illuminate the understanding of their function. We can envisage three important functions which may explain their roles in chromatin dynamics: (1) PML NBs may concentrate biochemical reactions. The biochemical environment within phase-separated PML NBs is different from the nucleoplasm and could serve to regulate (i) the kinetics of enzymatic reactions or (ii) the specificity of the modifications catalyzed. This is consistent with the described role of PML NBs as sumoylation hotspots, but could also apply for other modifications such as phosphorylation, acetylation, ubiquitination, or protein degradation. An example of the SUMOylation of a given client by UBC9 or of another client modification by a specific enzyme is shown. (2) PML NBs may buffer/sequester proteins via liquid-liquid phase separation of these client proteins. Increase in PML/client concentration may trigger accumulation of a given protein in PML NBs as a means to buffer the amount of the free protein in the nucleoplasm (as observed early for CBP for example). In addition, protein sequestration in PML NBs might affect their known activity as observed for DAXX. (3) PML NBs may help to organize specific nuclear domains, such as chromatin domains. PML NBs are interspersed in the active chromatin compartment and could potentially help to organize this compartment by pulling together genomic loci with similar transcriptional regulation. Of note, these three functions are not mutually exclusive and may serve altogether to regulate chromatin dynamics. Concentration of various factors in PML NBs together with specific genomic loci may help to catalyse specific reactions at given loci, as in the case of the ALT pathway for example (see Figure 3).
Figure 3.
Figure 3.
PML NBs directly regulate chromatin dynamics of DNA sequences found in the condensate. (A) Viral DNA-containing PML NBs (vDCP NBs) are specific PML NBs encasing the HSV-1 latent viral genome. Both H3.3 histone chaperone complexes (DAXX-ATRX and HIRA complexes) are found in these structures together with H3.3–H4. These complexes are essential for the H3.3 chromatinization of the virus, together with PML. H3.3 is decorated with the heterochromatin mark H3K9me3, which could be deposited by SETDB1 (question mark), a known client protein localizing constitutively in PML NBs. (B) PML NBs containing satellite DNA are found in the ICF syndrome in the form of giant PML NBs. These structures contain proteins organized in ordered concentric layers around the satellite DNA core, in the following order from the center: HP1 proteins, DAXX–ATRX complex, CBP/BLM/TOP3A, surrounded by a sphere of SUMO1/SP100 and then PML protein (concentric layers not shown). While the heterochromatin nature of the satellite DNA is atypical with absence of the constitutive H3K9me3 mark despite HP1 presence, the presence of γH2A.X in some giant PML NBs (25%) nevertheless suggests that satellite DNA is associated with chromatin inside PML NBs. Of note, normal PML NBs can also contain satellite DNA in G2 phase. PML NBs-containing satellite DNA may help remodelling and maintenance of the heterochromatin structure present at late-replicating satellite DNA. (C) ALT-associated PML NBs (APBs) are a hallmark of the ALT pathway. Here we only focus on the chromatin dynamics in APBs, and neither display the numerous repair factors present in APBs nor the mechanisms involved in ALT. Telomeric DNA localizes within PML NBs together with specific chromatin-related factors such as SETDB1, ASF1, or HIRA. Recent data suggest that telomeric DNA repeats are more compact, with higher levels of H3K9me3 deposited by SETDB1 (i), and bound less TRF2 in ABPs than regular telomeres (ii), which would cause telomeric deprotection and promote telomeric recombination. Increase in TERRA transcription (orange lines) is also observed (iii) and fuels the ALT process by increasing RNA:DNA hybrids (iv) and thus replicative stress. Depletion of the histone chaperone Asf1 promotes histone management dysfunction during telomeric replication and is sufficient to trigger ALT (v).
Figure 4.
Figure 4.
Role of PML NBs in transcriptional regulation. PML NBs has a dual effect on gene expression both facilitating or repressing expression of specific genes. (A) PML NBs regulates transcription through specific modifications of transcription factors or by modulating the availability of transcription factors or chromatin-related factors. (i) Upon Ras-induced senescence entry, p53 localizes in PML NBs which promotes its phosphorylation on serine 15 (not shown) as well as its acetylation on lysine 382 by CBP or MOZ, which may be counteracted by SIRT1. These PML-dependent modifications are required for p53 transactivation activity. TIP60 SUMO-dependent relocalization in PML NBs upon UV damage may also participate in p53 recruitment and stabilization (dashed arrow), thus favoring its transactivation activity. Oxidative stress can also trigger PML-dependent p53 activation conveying the ROS response (237). (ii) PML NBs can regulate proteins levels by SUMO-dependent poly-ubiquitination by RNF4 and subsequent proteasome-mediated degradation as observed for PML-RARα, or (iii) by caspase degradation as observed for SATB1. (iv) In senescence, PML NBs concentrate Protein Phosphatase 1 alpha (PP1α) together with Rb preventing its CDK-dependent phosphorylation and thus inhibiting E2F which remains sequestered in PML NBs and cannot activate cell-cycle promoting genes. (v) The DAXX histone chaperone brings new H3.3-H4 dimers within PML NBs but may then be sequestered preventing the transcriptional repression of its target genes such as Glucocorticoid receptor (GR) target genes. HDAC7 may also be sequestered to prevent repression of MEF2 target genes. (vi) The role for HIRA complex localization in PML NBs remains more enigmatic (question mark). (B) PML NBs could also participate in establishing chromatin domains that are either permissive or refractory to transcription. (i) Interaction between SATB1 and PML is essential to establish a specific chromatin-loop structure at the MHC class I locus and may serve to regulate transcriptional activity of genes within this locus. (ii) PML NBs can also provide a transcriptionally-permissive chromatin environment to neighboring loci (dashed green circle). In particular, binding to the short arm of the Y-chromosome (region YS300) to PML NBs allows anchoring of specific Y-linked gene promoters that are located away from this region (dashed line). PML NBs allow the maintenance of their transcriptional activity by excluding DNMT3A and preventing DNA methylation on these proximal promoters. Specific transcription factors or chromatin-related factors located in PML NBs (orange factor) could also contribute to gene expression in these chromatin domains. (iii) On the contrary, PML NBs may help to concentrate HP1 proteins on specific loci, possibly through phase separation of heterochromatin (dashed red circle), to promote repression of genes such as E2F target genes. SETDB1 may also participate in creating a repressing heterochromatin environment by depositing H3K9me3 on gene promoters such as for the Id2 gene. However, it remains to be determined whether these repressed loci are found in vicinity of PML NBs (question mark).
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