A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain

Abstract

Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721-725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719-721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.

Figure 1.

Localization of EYFP-SUV39H1 in mouse NIH3T3 as well as in human U2OS cells. (A) After transfection, EYFP-SUV39H1 localizes in NIH3T3 cells to distinct nuclear regions that are also visible by DAPI staining. In U2OS cells, EYFP-SUV39H1 also localizes at sites that are stained by DAPI, but these areas are less well defined. (B) NIH3T3 cells transfected with EYFP-SUV39H1 were labeled with antibodies specific for trimethylated H3K9 and HP1α. (C) Human U2OS cells transfected with EYFP-SUV39H1 were labeled with antibodies against centromeres. Single optical sections show the YFP-tagged protein, centromere labeling, and an overlay. Line scans (diagonal lines through the images) show the local intensity distributions of the EYFP fusion protein in yellow and of the centromere labeling in red. Bars, 10 μm.

Figure 2.

EYFP-SUV39H1 expression rescues the phenotype of Suv39h dn PMEFs. Suv39h dn and wild-type PMEFs transfected with EYFP-SUV39H1 were labeled with antibodies against trimethylated H3K9 (A) or HP1β (B). In nontransfected Suv39h dn PMEFs, a little trimethylated H3K9 is present throughout the nucleus (A, arrows). Transfected cells show an increase in trimethylated H3K9 that is localized to heterochromatic areas (A, middle and bottom). This localization is comparable to wild-type cells (A, top). Bars, 5 μm.

Figure 3.

FRAP analysis of EYFP-SUV39H1 and EYFP-HP1β shows that SUV39H1 is a less dynamic component of heterochromatin. NIH3T3 cells were transfected with EYFP-SUV39H1 (A) or EYFP-HP1β (B). A heterochromatic area was selected and photobleached. Images were recorded just before bleaching and at different time intervals after bleaching. Arrows indicate the photobleached region. To illustrate the recovery of fluorescence more clearly, pseudocolor images of the bleached cells are shown. Fluorescent intensities range from blue (low) to red (high). (C) Relative fluorescence intensities are displayed in recovery curves. Fluorescence recovery of EYFP-SUV39H1 reached a plateau at ∼70% after 140 s. Fluorescence recovery of EYFP-HP1β reached a plateau at ∼95% after 60 s. The curves represent mean values from 37 and 24 cells, respectively. Error bars represent SD. (D) FRAP curves calculated after extended recovery periods of EYFP-SUV39H1 in heterochromatin of U2OS cells, of Suv39h dn PMEFs, and of NIH3T3 cells and in euchromatin of NIH3T3 cells. (E) FRAP curves obtained from two successive FRAP measurements. After fluorescence recovery after the first bleach, the same region was bleached for the second time, and the fluorescence recovery was measured. EYFP-SUV39H1 fully recovered after the second bleach to 70% of the initial amount of fluorescence measured before the first bleach.

Figure 4.

Mutant SUV39H1 constructs. Overview of the SUV39H1 deletion mutants fused to EYFP. The chromodomain is shown as a gray shaded box, and the SET domain is in black. The numbers refer to amino acid positions in the SUV39H1 protein.

Figure 5.

SUV39H1-ΔSET and SUV39H1-Nchromo are more concentrated at centromeres than SUV39H1. (A) Localization of the various SUV39H1 mutants in NIH3T3 cells. Bar, 10 μm. (B) Western blot showing expression of EYFP-SUV39H1 (lane 1), EYFP–SUV39H1-ΔSET (lane 2), and EYFP–SUV39H1-Nchromo (lane 3). Ponceau staining shows equal loading of the gel. (C) The top panel shows the distribution of EYFP–SUV39H1-ΔSET, centromeres, and DNA in a U2OS cell. The bottom panel shows a single optical section of a U2OS cell expressing EYFP–SUV39H1-ΔSET (left) and stained for centromeres (middle). Colocalization of the two is shown in the right image. The line scan (diagonal lines through the images) shows the local intensity distribution of EYFP–SUV39H1-ΔSET in yellow and centromere labeling in red. (D) Simultaneous detection of EYFP–SUV39H1-Nchromo (left) and centromeres (middle) in a U2OS cell. DNA is stained by DAPI (top right). The bottom panel shows a single optical section and a line scan (diagonal lines through the images) giving the local intensity distribution of EYFP–SUV39H1-Nchromo (yellow) and centromere staining (red).

Figure 6.

SUV39H1-ΔSET and SUV39H1-Nchromo have a higher mobility rate than full-length SUV39H1. After transfection of NIH3T3 cells with EYFP–SUV39H1-ΔSET (A) or EYFP–SUV39H1-Nchromo (B), a heterochromatic area was selected and photobleached. Images were recorded just before and at different time intervals after bleaching. Arrows indicate the photobleached areas. (C) The corresponding FRAP curves are plotted together with the FRAP curves for EYFP-SUV39H1 and EYFP-HP1β. These curves indicate that EYFP–SUV39H1-ΔSET and EYFP–SUV39H1-Nchromo are more dynamic than the full-length protein. The FRAP curves for the two mutant proteins represent means from 30 and 14 cells, respectively.

Figure 7.

Methyltranferase activity has no influence on SUV39H1 dynamics. NIH3T3 cells were transfected with EYFP–SUV39H1-H324L or with EYFP–SUV39H1-H320R. Heterochromatic areas were selected and photobleached. Images were recorded just before and at different time intervals after photobleaching. The corresponding FRAP curves are plotted together with the FRAP curve for EYFP-SUV39H1. The curves represent mean values from 28 and 25 cells, respectively.

Figure 8.

FRET measurements confirm the interaction between different autofluorescent fusion proteins. U2OS cells were cotransfected with ECFP-SUV39H1 and EYFP-HP1β (A), with ECFP–SUV39H1-ΔSET and EYFP-HP1β (B), or with ECFP–SUV39H1-ΔN89 and EYFP-HP1β (C). A CFP, YFP, and FRET image was acquired from all cells for spectral measurements, and a FLIM stack was collected to calculate fluorescent lifetimes. The CFP, YFP, and FRET images were first corrected for pixel shift and background. Then, the N_FRET values were calculated and displayed in pseudocolors (Nfret). Absence of FRET is indicated by blue as depicted in the scale. The fluorescent lifetime values are also displayed in pseudocolors (FLIM). Blue (as depicted in the scale) indicates a lifetime of 2.5 ns and an absence of FRET.

Figure 9.

DNA demethylation increases the dynamics of both SUV39H1 and SUV39H1-ΔSET but not that of HP1β. NIH3T3 cells were transfected with EYFP-SUV39H1, EYFP–SUV39H1-ΔSET, or with EYFP-HP1β and were treated with 5 μM 5-aza-C. After 48–52 h of treatment, a heterochromatic area was selected and photobleached. Images were recorded just before and at different time intervals after photobleaching. The corresponding FRAP curves are plotted together with the FRAP curves for EYFP-SUV39H1, EYFP–SUV39H1-ΔSET, and EYFP-HP1β obtained from nontreated cells. Curves represent the means from 18, 12, and 16 cells, respectively, and show that both EYFP-SUV39H1 and EYFP–SUV39H1-ΔSET become more dynamic after 5-aza-C treatment.

Figure 10.

Inhibition of histone deacetylase activity increases the dynamics of both SUV39H1 and HP1β. NIH3T3 cells were transfected with EYFP-SUV39H1 (A) or EYFP-HP1β (B) and treated with 50 ng/ml TSA. After 18–22 h of TSA treatment, a heterochromatic area was selected and photobleached. Images were recorded just before and at different time intervals after photobleaching. Arrows indicate the photobleached areas. (C) The corresponding FRAP curves are plotted together with the FRAP curves for EYFP-SUV39H1 and EYFP-HP1β obtained from nontreated cells. Curves represent means from 22 and 8 cells, respectively. (D) Western blots showing the levels of acetylation and trimethylation in nontreated (lane 1), TSA-treated (lane 2), and 5-aza-C–treated (lane 3) NIH3T3 cells. Ponceau staining shows equal protein loading.