Pericentromeric heterochromatin domains are maintained without accumulation of HP1

Abstract

The heterochromatin protein 1 (HP1) family is thought to be an important structural component of heterochromatin. HP1 proteins bind via their chromodomain to nucleosomes methylated at lysine 9 of histone H3 (H3K9me). To investigate the role of HP1 in maintaining heterochromatin structure, we used a dominant negative approach by expressing truncated HP1alpha or HP1beta proteins lacking a functional chromodomain. Expression of these truncated HP1 proteins individually or in combination resulted in a strong reduction of the accumulation of HP1alpha, HP1beta, and HP1gamma in pericentromeric heterochromatin domains in mouse 3T3 fibroblasts. The expression levels of HP1 did not change. The apparent displacement of HP1alpha, HP1beta, and HP1gamma from pericentromeric heterochromatin did not result in visible changes in the structure of pericentromeric heterochromatin domains, as visualized by DAPI staining and immunofluorescent labeling of H3K9me. Our results show that the accumulation of HP1alpha, HP1beta, and HP1gamma at pericentromeric heterochromatin domains is not required to maintain DAPI-stained pericentromeric heterochromatin domains and the methylated state of histone H3 at lysine 9 in such heterochromatin domains.

Figure 1.

Distribution of transfected full-length HP1α-flag and HP1β-flag in mouse fibroblasts. Cells transfected with HP1α-flag (A) and HP1β-flag (B) were fluorescently immunolabeled 24 h after transfection. The red signal (mouse anti-flag) shows the distribution of transfected HP1α-flag (A) and HP1β-flag (B). The cyan channel shows the DAPI staining. Bars, 2 μm. Individual midnuclear optical sections are shown.

Figure 2.

Spatial distribution of transfected truncated HP1, endogenous HP1, and DAPI-stained heterochromatin domains. Cells transfected with HP1α-Δ(2-39)-flag (A and B) and HP1β-Δ(2-40)-flag (C–E) were fluorescently labeled 24 h after transfection. The red signal (Maflag in A–D and Raflag in E) shows the transfected HP1α-Δ(2-39)-flag (A and B) and HP1β-Δ(2-40)-flag (C–E). The green signal shows the distribution of endogenous HP1α, HP1β, and HP1γ after labeling with RaHP1α (A and C), RaaHP1β (B and D), and MaHP1γ (E). The cyan signal shows the DAPI staining. On the right, intensity profiles are shown of endogenous HP1α, HP1β, and HP1γ; green line) and DAPI stain (cyan line) along the lines shown in the DAPI images. A1, B1, C1, D1, and E1 are line scans in control cells, and A2, B2, C2, D2, and E2 in transfected cells. The x-axis shows distance in μm; the y-axis represents signal intensity in arbitrary units. Individual midnuclear optical sections are shown.

Figure 3.

Spatial distribution of EGFP-tagged HP1β-expressing cells transfected with mCherry-tagged HP1β-Δ(2-40). Cells stably expressing EGFP-tagged HP1β show that HP1 is accumulating in pericentromeric heterochromatin domains (A). EGFP-tagged HP1β expressing cells were transfected with mCherry-tagged HP1β-Δ(2-40) (B and C). The green signal shows the EGFP-tagged HP1β (B). The red signal shows the transfected mCherry-tagged HP1β-Δ(2-40) (C). The fluorescence intensity of EGFP-HP1β in cells trasfected with mCherry-tagged HP1β-Δ(2-40) was compared with nontransfected cells. A histogram of the Gaussian distribution of the EGFP-HP1β fluorescence intensity in transfected (E) and nontransfected cells (D) is shown. x-axis, the average fluorescence intensity in arbitrary units; y-axis, the number of cells.

Figure 4.

Quantitative analysis of the spatial distribution of transfected truncated HP1, endogenous HP1, and DAPI-stained heterochromatin domains. Maximum intensity projections of the imaged cells (see Supplementary Figure S1) were generated. Average signal intensity was measured for every channel over circular areas of 1.5 μm². These circles were placed over five DAPI dense chromatin regions and five non-DAPI dense chromatin regions in transfected and nontransfected control cells (B). The ratio between cells transfected with HP1α-Δ(2-39)-flag or HP1β-Δ(2-40)-flag versus not transfected control cells, for the DAPI signal (black bars) as well as for the endogenous HP1 signal (gray bars) was calculated. This was done in individual images as the ratio of the differences between average intensity in DAPI dense chromatin regions (HC) and non-DAPI dense euchromatic chromatin regions (EC) in transfected (dn) and nontransfected cells (wt): (HC − EC)_dn/(HC − EC)_wt (A). Error bars, SE between the different images.

Figure 5.

Spatial distribution of H3K9me in cells transfected with truncated HP1α and HP1β. Cells transfected with HP1α-Δ(2-39)-flag (A and B) and HP1β-Δ(2-40)-flag (C–E) were fluorescently labeled 24 h after transfection. The red signal (mouse-anti-flag) shows the transfected HP1α-Δ(2-39)-flag (A and B) and HP1β-Δ(2-40)-flag (C–E). The green signal shows distribution of H3K9me detected by either RaMeH3K9me3 (A and C), RaH3K9mebranched (B and D), or RaH3K9me2 (D). The cyan signal represents DAPI staining. Bars, 2 μm. Individual midnuclear optical sections are shown.