ATRX interacts with H3.3 in maintaining telomere structural integrity in pluripotent embryonic stem cells

Abstract

ATRX (alpha thalassemia/mental retardation syndrome X-linked) belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Besides the ATPase/helicase domain at its C terminus, it contains a PHD-like zinc finger at the N terminus. Mutations in the ATRX gene are associated with X-linked mental retardation (XLMR) often accompanied by alpha thalassemia (ATRX syndrome). Although ATRX has been postulated to be a transcriptional regulator, its precise roles remain undefined. We demonstrate ATRX localization at the telomeres in interphase mouse embryonic stem (ES) cells in synchrony with the incorporation of H3.3 during telomere replication at S phase. Moreover, we found that chromobox homolog 5 (CBX5) (also known as heterochromatin protein 1 alpha, or HP1 alpha) is also present at the telomeres in ES cells. We show by coimmunoprecipitation that this localization is dependent on the association of ATRX with histone H3.3, and that mutating the K4 residue of H3.3 significantly diminishes ATRX and H3.3 interaction. RNAi-knockdown of ATRX induces a telomere-dysfunction phenotype and significantly reduces CBX5 enrichment at the telomeres. These findings suggest a novel function of ATRX, working in conjunction with H3.3 and CBX5, as a key regulator of ES-cell telomere chromatin.

Figure 1.

Enrichment of ATRX at telomeres in mouse ES cell lines, ES129.1 and W9.5. Two sources of antibodies against ATRX (H300 and D19) were used in immunofluorescence analysis. (A,B) In interphase ES129.1 and ES-W9.5 cells, ATRX (Aiii,Biii, green; H300) was found at the telomeres (arrows) and pericentric heterochromatin (for ATRX localization at pericentric heterochromatin, as indicated by centromeric CREST staining, see Supplemental Fig. S1). Telomeric localization of ATRX (Aiii,Biii, green) was confirmed by telomere FISH (Aii,Bii, red). (C) Immunofluorescence analysis using a second source of antibody also showed ATRX (ii, red; D19) enrichment at the telomeres (arrows) in interphase ES129.1 cells, as evident from its colocalization with TERF1 (iii, green).

Figure 2.

Cellular distribution of ATRX in mouse ES cell lines ES129.1 and W9.5 and in mouse EG cell lines EGRA2 and EGRA3. (A) In interphase ES129.1 cells, a triple-staining immunofluorescence analysis showed clear colocalization of ATRX (iv, green) with telomere FISH signals (iii, red), without any CREST signal (arrows). ATRX localization at pericentric heterochromatin was indicated by its close proximity to the centromeric CREST staining (ii, blue; arrowheads). (B,C) In interphase EGRA3 and EGRA2 cells, ATRX (Biii,Ciii, green) was similarly enriched at the telomeres (arrows). Telomeric localization of ATRX (Biii,Ciii, green) was confirmed by telomere FISH (Bii,Cii, red).

Figure 3.

Timing of telomeric loading of ATRX. We previously showed that MYC-H3.3 was loaded at the telomeres in interphase ES129.1 cells during S phase (Wong et al. 2009). Here, ES129.1 cells were similarly synchronized using a thymidine-induced G₁/S-block protocol (for FACS analysis, see Supplemental Fig. S3). (A–C) ATRX (iii, green) was present at the telomeres in cells from 2–6 h post-release from G₁/S block, as indicated by colocalization with telomere FISH signals (ii, red). (D) By mitosis (8 h post-release), ATRX (iii, green) completely delocalized from the telomeres (ii, red; indicated by telomere FISH signals) but remained at pericentric heterochromatin (arrowheads). (E) Enlarged images of the boxed areas shown in D, showing no ATRX signals at the telomeres (arrows) but strong localization of ATRX (iii, green) at the pericentric regions (arrowheads); note that the telocentric nature of the mouse chromosomes meant an inevitable display of the observed colocalization of telomeric FISH signals with the centromeres at this resolution. At 10 h post-release, the cells re-entered G₁ phase of the cell cycle (data not shown).

Figure 4.

Cellular localization of MYC-H3.3 and ATRX in differentiated ES cells. (A,B) MYC-H3.3 construct was transfected into ES129.1 cells and induced with 1 μM doxycycline for 24 h MYC-H3.3 (Aii, red) colocalized with TERF1 (Aiii, green) at the telomeres (examples shown by the arrows) in undifferentiated (0 day) interphase ES 129.1 cells. MYC-H3.3 and ATRX signals remained apparent at the telomeres after 6 and 9 d of induction (data not shown). However, no enrichment of MYC-H3.3 (Bii, red) was detected at the telomeres (indicated by TERF1 staining; Biii, green) 12 d following the induction of differentiation. (C,D) Likewise, ATRX association at the telomeres was also not noticeably affected by the induction of differentiation for 6 and 9 d (Ciii, green), but ATRX (Diii, green) completely delocalized from the telomeres after 12 d of induction of cellular differentiation.

Figure 5.

Colocalization of ATRX and MYC-H3.3. (A) ATRX (iii, green) clearly colocalized with MYC-H3.3 (ii, red) in undifferentiated (0 day) interphase ES129.1 cells. (B) The colocalization of ATRX (iii, green) with MYC-H3.3 (ii, red) remained prominent after 6 d of differentiation. (C) However, after 12 d of differentiation, the colocalization of ATRX (iii, green) with MYC-H3.3 (ii, red) was greatly reduced. Occasionally, strong ATRX signals were detected in the 6- and 12-d cells, but these signals were not colocalized with the telomeres (Biii,Ciii, green; arrowheads).

Figure 6.

The effects of H3.3 lysine-4 mutation on its interaction with ATRX by immunofluorescence and immunoprecipitation/Western blot analysis. (A) As a positive control, we showed a clear colocalization of MYC-H3.3 (ii, red) with ATRX (iii, green) in ES129.1 cells. (B,C) The mutation of K4 (lysine to alanine) residue was introduced by site-directed PCR mutagenesis. The MYC-H3.3mutK4 plasmid DNA was transiently transfected into mouse ES129.1 cells. The expression of MYC-H3.3mutK4 (Biii, green; indicated by arrows) in ES129.1 cells (cell on the right) did not affect its telomeric localization (as indicated by telomere FISH signals; Bii, red) but resulted in a significant reduction in the association of ATRX (Ciii, green) with MYC-H3.3 (Cii, red; indicated by arrows) at telomeres compared with cells not expressing MYC-H3.3mutK4 (cell on the left). (D) In ES129.1 cells expressing either MYC-H3.3wtK4 or MYC-H3.3mutK4, the signal intensities of MYC-H3.3wtK4/ATRX and MYC-H3.3mutK4/ATRX were quantitated. For each cell, the intensities for five sets of colocalizing MYC-H3.3wtK4/ATRX and MYC-H3.3mutK4/ATRX signals were measured, and the average intensities were determined. In total, 25 cells were assessed (Supplemental Table S3). As shown in the scattered plot, in ES cells expressing MYC-H3.3wtK4, ATRX signal intensity remained high regardless of MYC-H3.3wtK4 signal intensity, whereas, an inverse relationship between the expression level of MYC-H3.3mutK4 and ATRX signal intensity was observed, suggesting that MYC-H3.3mutK4 expression affects ATRX association. (E) Immunoprecipitation with anti-ATRX antiserum pulled down MYC-H3.3 in ES129.1 cells expressing MYC-H3.3 (lane 2) but failed to pull down MYC-H3.3mutK4 in ES129.1 cells expressing MYC-H3.3mutK4 (lane 5). As a positive control, immunoprecipitation with anti-MYC antiserum also pulled down MYC-H3.3 (lane 3) and MYC-H3.3mutK4 (lane 6). The two bands above MYC-H3.3 in lanes 3 and 6 are heavy and light chains of the anti-MYC antiserum (clear arrowheads).

Figure 7.

Effect of ATRX RNAi-knockdown on telomere integrity in mouse ES cells. (A) Western blot analysis of ES129.1 cells transfected with ATRX-specific RNAi oligonucleotide sets (set 3 and sets 1–3 from Invitrogen) using anti-ATRX and anti-beta-tubulin antisera. (i) Data are presented in histograms by normalization of the intensity of ATRX levels against the intensity of beta-tubulin levels. (ii) ATRX RNAi-depleted cells showed a significant reduction of ∼90% in ATRX protein level (arrows; lanes 3,4) 48 h after the transfection, compared with nontransfected cells (lane 1) and cells transfected with scrambled control RNAi oligonucleotides (lane 2). The equal loading of protein was achieved by normalization against the beta-tubulin level. (B) Induction of TIFs by ATRX-inhibition using ATRX RNAi oligonucleotide (set 3 and sets 1–3 from Invitrogen and set 2 from Ambion, respectively) for 48 h. Data are presented in histograms by subgrouping the cells according to the number of TIFs per cell (less than five 5 TIFs, five to nine TIFs, 10 to 14 TIFs, and more than 14 TIFs). A normal cell can contain one to two TIFs on average; thus, a threshold of four or more TIFs was used, as described in other studies (Hockemeyer et al. 2005). When transfected with ATRX-RNAi oligonucleotides, the number of cells with five or more TIFs (85 cells were counted for each experiment) by three- to fourfold (increased from 9.41% to as high as 29.8%–34.90%, with an average increase of 20.39%–25.49%). In this study, we only counted the number of TIFs in interphase ES129.1 cells although in some metaphase cells, TIFs were also present at the telomeres following RNAi-depletion of ATRX. We also performed RNAi-knockdown using ATRX-specific RNAi oligonucleotide sets purchased from Ambion, showing a significant reduction of ∼80% in ATRX protein level and a fivefold increase in the number of cells with five or more TIFs 48 h after the transfection (see Fig. S7). (C–E) Immunofluorescence analysis of ES129.1 cells subjected to 48-h knockdown with either control (C) or ATRX-specific (D,E) RNAi-duplex oligonuclotides using anti-gamma-H2AFX (Cii,Eii; green) antiserum. Increased number of TIFs was detected at telomeres (indicated by telomere FISH analysis; Diii,Eiii) in cells depleted of endogenous ATRX (arrowheads show some examples of TIFs).

Figure 8.

Colocalization of CBX5 with ATRX at the telomeres in ES cells. (A) A triple staining/immunofluorescence analysis showing colocalization of CBX5 (iv, blue) with ATRX (ii, green) at the pericentric heterochromatin (arrowheads) and telomeres (iii, red; indicated by telomere FISH). (B,C) CBX5 (Biii, red) colocalized with TERF1 (Bii, green) at the telomeres (examples shown by the arrows) in undifferentiated (0 day) interphase ES 129.1 cells. CBX5 signal remained apparent at the telomeres after 6 and 9 d of induction (data not shown). However, CBX5 localization (Ciii, red) at the telomeres (Cii, green) was greatly decreased 12 d following the induction of differentiation. (D) In somatic cells, e.g., NIH3T3, CBX5 (iii, red) localized mainly at pericentric heterochromation, but its association with the telomeres (arrowheads; ii, green) was below detection.

Figure 9.

Effect of ATRX RNAi-knockdown on telomeric association of CBX5 in mouse ES cells. (A–C) ES129.1 cells transfected with scramble RNAi oligonucleotide (A) and ATRX-specific RNAi oligonucleotide sets (B–C; set 3 and sets 1–3 [Invitrogen]) for a period of 48 h. Transfection of ES129.1 cells with scrambled RNAi oligonucleotide did not affect localization of CBX5 (Aiii, red) at the telomeres (arrows), as indicated by costaining with TERF1 (Aii, green). However, RNAi depletion of ATRX with RNAi oligonucleotide sets, set 3 (B) and sets 1–3 (C) led to a significant dissociation of CBX5 from the telomeres (arrowheads) in ES129.1 cells.