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. 2012 May-Jun;3(3):276-85.
doi: 10.4161/nucl.20180. Epub 2012 May 1.

Dualistic function of Daxx at centromeric and pericentromeric heterochromatin in normal and stress conditions

Viacheslav M Morozov  1 , Ekaterina V Gavrilova, Vasily V Ogryzko, Alexander M Ishov
Affiliations

Dualistic function of Daxx at centromeric and pericentromeric heterochromatin in normal and stress conditions

Viacheslav M Morozov et al. Nucleus. 2012 May-Jun.

Abstract

Nuclear structures ND10/PML NBs are linked to multiple processes, including the maintenance of intranuclear homeostasis by sequestering proteins into "nuclear depot." This function presumes release of proteins from PML NBs and their redistribution to the alternative, supposedly "active" locations, in response to the external stress application. To further investigate this nuclear depot function, we focused on the intranuclear distribution of protein Daxx that in normal conditions is mainly accumulated at PML NBs, and has a minor association with centromeres and pericentromeres (CEN/periCEN). Here we report that application of physiological Heat Shock (HS) changes this balance forcing very robust and reversible accumulation of Daxx on CEN/periCEN heterochromatin. Heterochromatin architecture is essential for the proper orchestration of nuclear processes, while transcription from this part of genome is required for its maintenance. To understand functional consequences of Daxx deposition at CEN/periCEN, we tested for Daxx-dependency of heterochromatin transcription. Depletion of Daxx reduces accumulation of CEN RNA in normal conditions and periCEN RNA after HS application. Searching for the mechanism of Daxx-dependent regulation of heterochromatin transcription, we found that depletion of Daxx decreases incorporation of transcription-associated histone H3 variant, H3.3, into both CEN and periCEN. Surprisingly, HS-induced deposition of Daxx does not further elevate incorporation of H3.3 into CEN/periCEN that remained steady during stress and recovery. Instead, depletion of Daxx leads to HS-induced changes in the balance of epigenetic modifications at heterochromatin, most dramatically elevating levels of active H3K4Me2 modification at periCEN. We propose dualistic function of Daxx-containing complexes at CEN/periCEN: (1) regulation of H3.3 loading in normal conditions and (2) protection of epigenetic status upon stress-induced accumulation, thus collectively guarding epigenetic identity of CEN/periCEN heterochromatin.

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Figures

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Figure 1. Daxx accumulation at CEN/periCEN. HEp2 cells were fixed at 37°C or exposed to 42°C for 1 h and either fixed immediately or recovered at 37°C for 1 h or 2 h. Daxx is mostly associated with PML NBs (visualized by anti-PML ab’s) in control conditions (A); it forms additional domains after HS and after 1 h recovery (B and C) and is mostly returned to pre-stress localization after 2 h recovery (D). Daxx co-localizes with centromeres (visualized by CREST human autoimmune ab’s) in some cells at 37°C (E, inset 2); this co-localization is obvious in most cells after HS and 1 h recovery (F and G, inset 1), and is decreased after 2 h recovery (H); in addition, Daxx accumulates juxtaposed to centromeres upon recovery (G, inset 2). HSF1 is nuclear homogenous in control conditions (I) and forms stress bodies (SBs) upon HS; Daxx is adjacent to SBs after HS/1 h recovery (insets L–K), and co-localizes with majority of SBs at 1 h recovery (K, insets 2 and 3). At 2 h recovery, Daxx is associated with some SB (L; inset 1 high association, inset 2 low association). (M) Colocalization analysis (Pearson’s correlation coefficient upon HS and recovery) has been used to quantify the degree of association for Daxx/PML and Daxx/centromeres (CREST). Data are the means of three experiments, and the standard deviation is shown. (N) ChIP analysis of Daxx association with CEN/periCEN in HEp2 cells stably expressing FLAG-HA-Daxx. Daxx association with CEN (α-satellite) was minor at 37°C, was elevated ~10-fold upon HS (p < 0.001), was still high, but reduced, at 1 h of recovery and returned to pre-stress levels at 2 h and 3 h. Daxx association with periCEN (HS 3-9) was minimal at 37°C, was elevated upon HS (p < 0.001) and 1 h of recovery, and reduced to almost pre-stress levels at 3 h of recovery.
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Figure 2. Daxx-dependent expression of CEN/periCEN upon HS and recovery. RNA was purified from control-(CTL) and Daxx-depleted HEp2 cells at 37°C, immediately after HS at 42°C for 1 h or after recovery at 37°C for 1–12 h; qPCR analysis of α-satellite repeats transcripts (CEN, left) and HS3-9 transcripts (periCEN, right). Transcript levels were normalized to GAPDH expression levels. Bars represent the mean between replicates (mean ± SD).
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Figure 3. Association of histone H3.3 and H3.1 with CEN/periCEN. Control- or Daxx-depleted HEp2 cells expressing FLAG/HA-tagged H3.3 or H3.1 were used for FLAG-ChIP in normal condition (37°C) or after 1 h HS/1 h recovery. qPCR data for H3.3 and 3.1 association with α-satellite repeats (CEN) or HS3-9 (periCEN) are presented as fold enrichment IP over input; cells expressing FLAG/HA without an insert were used as negative control (pOZ). Bars represent the mean between replicates (mean ± SD).
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Figure 4. Analysis of epigenetic modifications on CEN/periCEN. ChIP for active (histone H3K4Me2) and repressive (histone H3K9Me3) chromatin marker at α-satellite (CEN) and HS3-9 (periCEN) TRs at normal conditions (37°C) and in post-stress time points (1 h and 12 h recovery). Bars represent the mean between replicates (mean ± SD).
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