Mode of SUV420H2 heterochromatin localization through multiple HP1 binding motifs in the heterochromatic targeting module

Abstract

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

Keywords: H3K9me3; H4K20me3; HP1; SUV420H2; chromatin; epigenetics; heterochromatin.

FIGURE 1

Human SUV420H2 contains three HP1 binding sites within HTM. (a) Schematic representation of SUV420H2 domains, including the amino acid sequence of HTM. The N‐ and C‐terminal heterochromatic targeting regions (aa 352–380 and 389–410) are labeled as HTM‐N and HTM‐C, respectively. Val residues in HP1 binding motifs are indicated in red. (b–e) Analysis of the impact of Val to Asp mutations in HP1‐binding PxVxL‐like motifs on heterochromatin localization. sfGFP‐tagged HTM‐N (b), HTM‐C (d), and full HTM (e), along with their mutants, were expressed in A9 cells. Heterochromatin accumulation of each sfGFP‐tagged protein is represented as heterochromatin enrichment ratio relative to the nucleus (c). (b, d, and e) Single confocal sections of typical nuclei. (c) Boxplots. Center lines represent the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th to 75th percentiles; × denote the means; and individual data points are shown as dots. Median values and number of analyzed nuclei are indicated above and to the right of box plots, respectively. Scale bars: 10 μm.

FIGURE 2

HTM‐N and HTM‐C interaction with HP1 through their PxVxL‐like motifs. (a) Analysis of HeLa cells expressing sfGFP, HTM‐N‐sfGFP, and HTM‐N V354D‐sfGFP (in which Val354 is substituted with Asp). Cells were treated with a buffer containing 0.1% Triton X‐100 and 1 M NaCl to extract most chromatin‐binding proteins (whole cell fractions; lanes 1–3). Post‐centrifugation, the supernatants (1 M NaCl extract; lanes 4–6) were separated from the pellet (1 M NaCl pellet; lanes 13–15) and utilized as inputs for immunoprecipitation using anti‐GFP beads. The unbound fractions (Unbound; lanes 7–9) and the immunoprecipitated materials (IP; 40× concentrated compared to the input; lanes 10–12) were processed. Protein samples were then separated on SDS‐polyacrylamide gels, transferred to membranes, and probed with the indicated antibodies. (b) Extracts from HeLa cells expressing sfGFP, HTM‐C‐sfGFP, and HTM‐C V402D‐V404D‐sfGFP were prepared, immunoprecipitated, and immunoblotted, as described in (a). The full blots are shown in Supplementary Figure S8.

FIGURE 3

Functions of the linker region between two HP1 binding motifs in HTM‐N. (a) The evolutionally conserved His and Cys residues are critical for HTM‐N heterochromatin targeting. Single amino acid substitution mutants of HTM‐N (H357A, C362V, and C366V) were expressed in A9 cells and their localization was analyzed using confocal microscopy. (b) The heterochromatic accumulation of HTM‐N is weakened by the insertion of a flexible linker between the two HP1 binding motifs and the deletion of the second motif. Single confocal sections of typical nuclei are shown, along with quantitative data as in Figure 1. The averages of enrichment ratios and the numbers of analyzed nuclei are indicated above and below box plots, respectively. p‐values by Tukey–Kamer test are indicated. Scale bars: 10 μm.

FIGURE 4

Expression of HTM‐sfGFP and HTM‐N‐sfGFP facilitates HP1β accumulation in H3K9me3‐enriched heterochromatin. (a and b) Effects of SUV420H2 HTM expression on HP1β accumulation in H3K9me3‐enriched heterochromatin. HeLa cells expressing HTM, HTM‐N, HTM‐C, and HTM V374D, each tagged with sfGFP, were stained with specific antibodies directed against H3K9me3 and HP1β. DNA was counterstained with Hoechst 33342. (a) Single confocal sections of typical nuclei. See Figure S4 for larger fields of view containing multiple nuclei. (b) Quantification of HP1β on H3K9me3‐marked heterochromatin. Using images like those shown in (a), the average HP1β intensities in the top 10% of H3K9me3 highest intensity pixels were measured and normalized to the mean intensities in entire nuclei. HP1β enrichments in H3K9me3‐enriched heterochromatin in individual nuclei from a single experiment are shown as dot and box plots on the left. The numbers of nuclei (N) analyzed are indicated above the box plots. Mean values of individual experiments in biological triplicates are shown on the right. Between 17 and 37 nuclei were analyzed for each mutant in single experiments. p‐values obtained from Student's t‐test (paired, two‐tailed) are indicated. (c and d) Co‐expression of sfCherry‐HP1α with HTM, HTM‐N, HTM‐C, and HTM V374D, each tagged with sfGFP. (c) Representative images of sfCherry‐HP1α in live cells when co‐expressed with HTM and its mutants are displayed. (d) Fluorescence recovery after photobleaching. During time‐lapse imaging, an area containing sfCherry‐HP1α focus (yellow circle) was bleached (top panels). The graphs below show the relative fluorescence intensities of sfCherry‐HP1α in the bleached area, normalized by those before bleaching (mean ± s.e.m.). The total number of cells (N) analyzed from two independent experiments are shown. Scale bars: 10 μm.

FIGURE 5

Endogenous SUV420H2 subtly contributes to HP1β locking. (a and b) Slight decrease of HP1β enrichments in H3K9me3‐enriched heterochromatin in SUV420H1/H2 double knockout (DKO) HeLa cells. Parental and three DKO HeLa cell lines were stained with antibodies specific to H4K20me3, H3K9me3, and HP1β. (a) Single confocal images of typical nuclei are shown. See Figure S5A for larger fields of view containing multiple nuclei. (b) Box plots. Average HP1β intensities in the top 10% of H3K9me3 highest intensity pixels were measured and normalized using the mean intensities in entire nuclei. HP1β enrichment in H3K9me3‐enriched heterochromatin in individual nuclei from a single experiment is represented as dot and box plots on the left, with the numbers of nuclei (N) analyzed indicated above. Mean values of individual experiments in biological quadruplicates are shown on the right. p‐values obtained from Dunnett's test are indicated. Between 83 and 136 nuclei were analyzed for each cell line in single experiment. (c and d) The effect of HTM‐sfGFP on HP1β accumulation in H3K9me3‐enriched heterochromatin in DKO cells. Parental HeLa and DKO (clone B6) cells transfected with HTM‐sfGFP were stained with antibodies specific to H3K9me3 and HP1β. (c) Single confocal images of typical nuclei. See Figure S5B for larger fields of view containing multiple nuclei. (d) Box plots as described in (b). Experiments were performed in triplicate with 52–156 cells analyzed in each line in single experiments. p‐values obtained from Tukey–Kramer test are indicated. Scale bars: 10 μm.

FIGURE 6

Visualizing HP1‐bound heterochromatin in living cells using HTM V374D‐sfGFP. (a and b) Distribution of HTM‐sfGFP, HTM V374D‐sfGFP, sfGFP‐HP1α, HP1α‐sfGFP in interphase nuclei of HeLa cells. H2B‐Halo was co‐expressed to visualize global distribution of chromatin. (a) Confocal images. Arrows indicate the accumulation of sfGFP‐tagged HP1 in small foci devoid of H2B‐Halo. (b) The contrast (coefficient of variation) of sfGFP‐tagged proteins in HeLa cells. Coefficient variations of the pixel intensities in single nuclei from a single experiment are shown in box plots on the left. The numbers of analyzed nuclei are indicated above the plots. Mean values from individual experiments in biological triplicates are shown on the right. p‐values obtained from Dunnett's test are indicated. (c) Distribution of HTM‐sfGFP, HTM V374D‐sfGFP, HP1α‐sfGFP, sfGFP‐HP1α in mitotic cells, with H2B‐Halo co‐expressed to visualize chromosomes. (d) Distribution of HTM‐sfGFP and HTM V374D‐sfGFP with co‐expressed sfCherry‐HP1α in mitotic cells. High‐power views of boxed area are shown in insets. (e) Dynamics of HP1‐enriched heterochromatin during DNA replication in the late S phase. Yellow arrowheads indicate the same foci, with high‐power views shown in insets. Scale bars: 10 μm.