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First published online 13 July 2004
doi: 10.1242/jcs.01230

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Heterochromatin and ND10 are cell-cycle regulated and phosphorylation-dependent alternate nuclear sites of the transcription repressor Daxx and SWI/SNF protein ATRX

Alexander M. Ishov^*, Olga V. Vladimirova and Gerd G. Maul

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA

View larger version (58K): [in a new window]   Fig. 1. Daxx is a chromatin-associated protein. (a) MPEF double labeled for PML (red) and Daxx (green). Both proteins localize at ND10, and Daxx also accumulates in a different shaped domain (upper right nucleus). (b) MPEF double labeled for HP1 and Daxx. Daxx localizes with HP1 at heterochromatin (middle right). (c) In PML–/– cells, Daxx accumulates in heterochromatin-like domains that localize with HP1 (d). (e) Western blot of intact MPEF cells (lane 1), cytoplasm (lane 2), nuclear insoluble fractions (n.p.) digested by micrococcal nuclease (MNase) (lane 3) and control (lane 4). Treatment with MNase resulted in the complete solubilization of Daxx and HP1 (lane 3). Loads in lanes 1 and 2 correspond to 10% of cells used in lanes 3 and 4. The asterisk indicates SUMOylated PML.

View larger version (108K): [in a new window]   Fig. 2. Cell-cycle-dependent distribution of Daxx. (a-c) MPEF labeled for phospho-histone H3 and Daxx show that no Daxx accumulates at heterochromatin during G1, G2 or M phase. (d-g) MPEF pulsed with BrdU for 30 minutes and triple-labeled for BrdU, ND10 and Daxx show Daxx located at heterochromatin during late S-phase but not during middle S-phase. (h-j) MPEF pulsed with BrdU for 30 minutes, chased for 3 hours and double-labeled for BrdU and Daxx show Daxx in ND10-like domains but not in heterochromatin of the cell that was at late S-phase during the pulse, and on heterochromatin in cell that was at early S-phase during the pulse. (k-m) PML–/– cells pulsed with BrdU for 30 minutes and labeled for BrdU and Daxx show Daxx concentrated at heterochromatin only during late S-phase despite the absence of ND10.

View larger version (55K): [in a new window]   Fig. 3. ATRX localizes with Daxx at heterochromatin and at ND10. (a) MPEF cells triple-labeled for HP1, ATRX and ND10, and the respective color separations (b-d) show that ATRX localizes with heterochromatin and ND10. (e) MPEF cells triple-labeled for ATRX, Daxx and ND10 and the respective individual colors (f-h) shows that Daxx localizes with ATRX to ND10 in most cells and, in some cells, with heterochromatin-like domains (upper left).

View larger version (85K): [in a new window]   Fig. 4. Characterization of Daxx knockout (KO) embryos and cell line. (a) Embryos were collected at the indicated days after mating (E), genotyped for Daxx wild type (wt) and KO and photographed. At E8, Daxx–/– embryos were developmentally retarded and were prominently so by E10.5. At E11.5, Daxx–/– embryos started to dissolve and were completely disintegrated by E12.5 (inset in Daxx–/– E12.5 shows 40x magnification). Daxx+/– embryos did not differ developmentally from Daxx+/+ embryos. (b) Genotyping of Daxx–/–, Daxx+/– and Daxx+/+ cell lines by PCR indicating the presence of corresponding alleles, whereas controls (wt control, KO control, H2O) demonstrated the specificity of the signals. (c) Western-blot analysis of Daxx–/–, Daxx+/– and Daxx+/+ cell lines. Daxx-specific signal (arrow) was present in HEp2, Daxx+/+ and Daxx+/–, but not in Daxx–/– cells. Unspecific signal is marked by the asterisk. (d-g) Cells labeled for Daxx and PML demonstrate colocalization of Daxx and PML to ND10 in Daxx+/+ (d) and Daxx+/– (e), but not in Daxx–/– (f), cells. (g) Daxx–/– cells double-labeled for ATRX and PML show ATRX accumulated in heterochromatin but not at ND10.

View larger version (92K): [in a new window]   Fig. 5. Production and cell-cycle analysis of cells reconstituted by Daxx wild-type (wt) and truncation mutants. Schematic of constructs is given at the top. Colocalization of reconstituted Daxx wt with HP1 at condensed chromatin (a, upper and lower nuclei) and with PML at ND10 (d) resemble those of endogenous Daxx. (g) BrdU incorporation followed by labeling of BrdU and Daxx by HA antibody; reconstituted Daxx wt accumulated at heterochromatin only at late S-phase (lower right nucleus). (b) DaxxC localized with HP1 at heterochromatin (upper and left nuclei) and is homogeneously dispersed in other cells. (e) DaxxC does not accumulate at ND10. (h) DaxxC accumulation at late BrdU incorporation sites (lower right). (c) DaxxC does not accumulate with HP1 at heterochromatin but maintains exclusive PML/ND10 associated pattern (f) throughout the cell cycle, including cells with a late S-phase BrdU incorporation pattern (i, lower right).

View larger version (65K): [in a new window]   Fig. 6. Intranuclear distribution of ATRX in Daxx reconstituted cells. (a) Daxx wild-type (wt) reconstituted cells labeled for ATRX and PML show colocalization of the proteins at ND10 (yellow), indicating a Daxx requirement in ATRX recruitment to ND10. (d) The same cells stained with ATRX and HA show colocalization of ATRX with Daxx at ND10-like domains. (e) DaxxC accumulation at ND10-like domains but no recruitment of ATRX to ND10 (b). In cells reconstituted with DaxxC and double stained for ATRX and PML (c) or HA and ATRX (f), ATRX does not accumulate at ND10 but has exclusive accumulation in irregular domains (c) while localizing with HA-DaxxC in a subpopulation of cells at irregular heterochromatic domains (f, lower left and right cells). (g) Western-blot analysis of interaction between ATRX and Daxx. Nuclear extracts produced from Daxx–/– cell lines reconstituted with empty vector (lane pBabe), Daxx wt (lane pBabe wt), DaxxC (lane pBabeDaxxC) and DaxxC (lane pBabeDaxxC) were incubated with anti-FLAG antibody to immunoprecipitate Daxx (FLAG IP) and probed for the presence of ATRX. No ATRX-derived signal was detected in extracts from the empty vector and DaxxC-reconstituted cells, whereas the signal was present in immunoprecipitates of Daxx wt and DaxxC cell extracts. Corresponding input (20%) for the ATRX load is shown (right).

View larger version (69K): [in a new window]   Fig. 7. Cell-cycle-dependent intranuclear distribution of SSRP1. (a) Cells double-labeled for SSRP1 and PML show that SSRP1 does not accumulate at ND10 but is present at irregular domains in some cells (upper left and right cells). (b) Double-labeling for SSRP1 and HP1 reveals SSRP1 at heterochromatin in some cells (two cells at lower right). (c) Double-labeling for Daxx and SSRP1 demonstrates co-localization of the proteins in heterochromatic domains (upper middle cell). (d) Cells pulsed with BrdU and labeled for SSRP1 and BrdU show homogeneous nuclear distribution of SSRP1 in most cells but localization with the late BrdU incorporation pattern (S-late). (e) Cells labeled for SSRP1 and phosphorylated histone H3 shows accumulation of SSRP1 at heterochromatin at the beginning of histone H3 phosphorylation corresponding to late S-phase and early G2 phase (S/G2) but not late G2 phase (G2). (f) The phosphorylated histone H3 signal shown separately.

View larger version (88K): [in a new window]   Fig. 8. Requirement for cellular kinase activity in Daxx and SSRP1 accumulation at heterochromatin. Relative to control (a), Daxx after roscovitine treatment of cells (d) shows no heterochromatic accumulation but it remains localized with PML. (b,e) ATRX and PML remain colocalized after roscovitine treatment and ATRX still accumulates at heterochromatin. The colocalization of Daxx and SSRP1 in heterochromatin in control cells (c, upper left) is absent from roscovitine-treated cells (e). (g) MPEF cell extracts were prepared without treatment (control lane), and 1 hour after roscovitine treatment; untreated (lane control 1) or protein-phosphatase-treated (lane phosphatase). Although Daxx, PML and HP1 did not change mobility after roscovitine treatment, the new ATRX band (*) migrates similarly to ATRX after phosphatase treatment.

View larger version (25K): [in a new window]   Fig. 9. Timeline of the reconfiguration of a multiprotein complex at condensed chromatin. Proteins from different nuclear domains accumulate together at heterochromatin in an apparent ATRX-based complex during late S-phase. Signaling for this change is due to a shift in phosphorylation of the heterochromatin-based ATRX. This complex is resolved during G2 phase, when ATRX recruitment to ND10 is mediated by Daxx.

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