SUPPLEMENTARY INFORMATION Dynamic Phosphorylation of HP1 Regulates Mitotic Progression in Human Cells Supplementary Figures Supplementary Figure 1. NDR1 interacts with HP1. (a) Immunoprecipitation using HA antibody in cells expressing YFP vector or YFP-HP1α cotransfected with HA-NDR1. Note HA-NDR1 does not interact with YFP protein (detected by GFP immunoblot). (b) Immunoprecipitation using HA antibody in cells expressing HA-HP1 wild type or HA-HP1 -W174A with T7-NDR1. Note the interaction between T7-NDR1 and HA- HP1 -WT but not W174A mutant. (c) Contrast adjusted YFP-LacI-HP1 tethered cells. Note that in addition to the CLTon heterochromatic locus, HP1 signal is localized to heterochromatic regions within the nucleus. Scale bar represents 10 m. 1
Supplementary Figure 2. NDR kinase mediates the phosphorylation of HP1. (a) In vitro kinase assay using NDR1 kinase isolated using HA immunoprecipitation from HA-NDR1 expressing human HEK293 cells. GST or GST-HP1 WT or truncation mutants 1-66, 67-119, 1-119 and 120-191 were used as substrates. Note the robust phosphorylation of GST-HP1 containing the chromo (1-66) and the hinge domain (67-119). Coomassie stained gel is shown for the substrates. Eluate from un-transfected HEK293 cells containing nonspecifically HA-bound proteins was used for control kinase assay using GST-HP1 WT as a substrate; the signal obtained from this assay was used to normalize the signal obtained from wild type NDR1. (b). Phos-tag PAGE analysis of lysates expressing HA-NDR1 along with single point mutants in N-terminal region of HP1 (S11A; S12A; S13A; S14A); a combined N-terminal mutant (S11-14A); single point mutants in the hinge region (S92A; S93A; S95A; S97A); a combined 2
hinge mutant (S92,93,95,97A). (c) Phos-tag PAGE analysis of lysates expressing HA-NDR1 along with single point mutants in the hinge region (S92A; S93A; S95A; S97A) and the combination of all (hinge mutant). CIP represents lysates treated with calf intestinal phosphatase. (d) Phos-tag PAGE analysis of lysates from cells transfected with FLAG-HP1 and with (+) or without (-) HA-NDR1 kinase, cells were synchronized at mitosis using nocodazole. Quantitation of the relative levels of N-terminal and N- terminal+ hinge phosphorylation of HP1 is based on the immunoblot in d. 3
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Supplementary Figure 3. Hinge-specific phosphorylated form of HP1 localizes at kinetochores during early mitosis. (a) Immunofluorescence localization of hinge-specific phosphorylation of HP1 using S95 phospho-ab (red) along with centromere marker ANA-C (green) in human U2OS cells. Note the association of phosphorylated form of HP1 in the vicinity of centromeres in prophase and metaphase. Inset (5X) shows that the red signals are exterior to green, suggesting the presence of this modified form of HP1 at outer kinetochores. Scale bar represents 10 m. (b) Immunofluorescence localization using preimmune sera (prebleed) and the centromeric ANA-C antibody in U2OS cells. Centrosomes (arrowhead) were non-specific staining. Scale bar represents 10 m. (c) Immunofluorescence using peptide-blocked pser95 antibody in HeLa cells. Note that the centromeric specific signal is lost. Scale bar represents 10 m. (d) Immunofluorescence localization of hinge-specific phosphorylation of HP1 using S95 phospho-ab (green) and HP1 antibody (red) in human HeLa cells. Scale bar represents 10 m. (e, f) Co-localization of S95 phospho-ab (red) and outer kinetochore marker Mad1 (green) in prometaphase and metaphase cells. Note the kinetochore association of the hingespecific phosphorylated form of HP1 is reduced as the cells progressed from prometaphase to metaphase. Arrowhead represents non-specific staining at centrosomes. Scale bar represents 10 m. 6
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Supplementary Figure 4. Hinge-specific phosphorylation of HP1 is required for accurate chromosome alignment and mitotic progression. (a). Immunoblot analysis of NDR depleted cell lysates using shrna against both NDR1 and 2. Note that >75% knock-down of NDR was achieved without significant alteration of total cellular level of other HP1 isoforms. (b) Immunoprecipitation using T7 antibody in cells expressing T7-NDR2 and HA-NDR1. (c) Immunoprecipitation using T7 antibody in cells expressing T7-NDR1 and HA-NDR1. Note the interaction between NDR1 and NDR2. (d) Flow cytometry in shrna mediated NDR1/2 depleted cells demonstrated an increase in G2/M population. (e) Immunofluorescence analysis of NDR1/2 depleted cells revealed mitotic abnormalities. Scale bar represents 10 m. (f) An increase in prometaphase and a concomitant decrease in metaphase population were also associated with NDR1/2 depletion. Error bars represent SD of three independent experiments. (g) Immunofluorescence localization of hinge-specific phosphorylation of HP1 using S95 phospho-ab (red) along with centromere marker ANA-C (green) in human HeLa cells. Note the reduction of phosphorylated form of HP1 in the vicinity of centromeres in NDR1-depleted cells. Scale bar represents 10 m. 8
Supplementary Figure 5. Hinge-specific phosphorylation of HP1 facilitates HP1 and Sgo1 binding to mitotic centromeres. (a) Immunoblot analysis to demonstrate the efficiency of HP1 sirna (3 UTR-specific) treatment in cells expressing U2OS, YFP-HP1 -S95A and YFP-HP1 -S95E. (b) Localization of YFP-HP1 -S95A phospho mutant and YFP-HP1 -S95E on mitotic chromosome in cells lacking endogenous HP1. Scale bar represents 10 m. (c) Immunoprecipitation experiments demonstrate that HP1 phosphorylation mutants YFP-HP1 -S95A and YFP-HP1 -S95E dimerize with WT-HP1. (d) Hinge-specific phosphorylation of HP1 facilitates Sgo1 binding to mitotic centromeres. Depletion of HP1 from U2OS or from cells stably expressing YFP-HP1 -S95A or YFP-HP1 -S95E followed by immunofluorescence using Sgo1 antibody. Quantification of Sgo1 present at mitotic centromeres. Note the increased binding of Sgo1 to centromeres in HP1 -depleted YFP-HP1 -S95E expressing cells. The bar graphs for control sirna are identical to the one depicted in Figure 6e control. The control-sirna, NDR1-siRNA and HP1 -sirna experiments (one group) were conducted at the same time. Error bars represent SD of three independent experiments. Statistical significance was determined by Student s t- test.. Mean ± SD, *p<0.05, **p<0.01 and ***p<0.001. 9
Supplementary Figure 6. Uncropped scans of the most important western blots. Boxes highlight the lanes used in panels. 10