B Western blot of the p150caf-1 complex

Transcription

1 Loyola et al., Supplementary Figure 1 A MS data of the HP1α complex RIF1 SPT6 SMARCA4 POGZ CAF-1 p150 KAP-1 HP1α CAF-1 p150 tnasp Importin4 B Western blot of the p150caf-1 complex Input mock Unbound Eluate Input Flag-p150 Unbound Eluate p150 p60 p48 Asf1a Asf1b SKB1 CAF-1 p60 NDR Tubulin α/β CAF-1 p60 snasp RbAp48 Hat1 HP1α SetDB1 actin e-hp-1α e ASF1a ASF1b H4 coomassie matched peptides Coverage ( % ) RIF-1 POGZ CAF-1 p KAP CAF-1 p Tubulin α Tubulin β HP-1 α 13 52

2 Loyola et al., Supplementary Figure 2 A Silver staining of glycerol gradient Input glycerol gradient fractions CAF-1 p150 KAP1 CAF-1 p60 SKB1 HP1 α HP1γ B HMTase assay of glycerol gradient glycerol gradient fractions me H4me HMTase Flag-mock Flag-HP1α Flag-HP1α * HP1α Histone Coomassie Blue staining C HMTase assay of HP1a and.1 complexes * autorad coomassie

3 Loyola et al., Supplementary Figure 3 A SetDB1 knock-down sicontrol sisetdb1 SetDB1 Tubulin B Western blot of Histone Nuclear Extract Chromatin Extract sicontrol sicontrol sisetdb1 sisetdb1 K9me1 K9me1 Western blot Ponceau Staining

4 Loyola et al., Supplementary Figure 4 A Western blot of nucleosomal sicontrol sisetdb1 B Western blot of non-nucleosomal sicontrol sisetdb1 50% reduction K9me1/ ratio K9me1 40% reduction K9me1/ ratio K9me1 C Western blot of HP1α-enriched nucleosomes chromatin control/setdb1 sirna sicontrol sisetdb1 MNase Flag-IP 10% reduction K9me2/ ratio e-hp1α K9me2

5 Loyola et al., 2009 SUPPLEMENTARY MATERIAL SUPPLEMENTARY METHODS Primary Antibodies. We used the following antibodies for western blot: anti- Asf1a/b (Groth et al., 2005), anti-caf-1 p60 (Marheineke and Krude, 1998), antip150caf-1 (Novus Biologicals #NB A1), anti-flag (Sigma #F3165), (Abcam #ab1791), K9me1 (Upstate #07-450), K9me2 (Upstate #07-212), K9me3 (Upstate #07-442), anti-hp1α (Euromedex #2HP-2G9-AS), KAP-1 (Abcam #ab10484), anti-mam (Want et al., 2003), anti-mbd1 (Imgenex #IMG- 306), anti-p48 (Abcam #ab1766), anti-setdb1 (Schultz et al., 2002), anti-tubulin (Sigma #T6074). We used the following antibodies for IF: anti-pcna (Dako #M879), anti-setdb1 (Schultz et al., 2002). Secondary Antibodies. We used the following antibodies for western blot: antimouse conjugated with peroxidase (Rockland # ) and anti-rabbit conjugated with peroxidase (Rockland # ). We used the following antibodies for IF: anti-mouse Alexa Fluor 488 (Molecular Probes #A21202) and anti-rabbit Alexa Fluor 594 (Molecular Probes #A21207). Westernblot. We harvested cells to prepare cytosolic, nuclear and chromatin extract (Dignam et al., 1983). We resolved histones on 15% SDS-PAGE and transferred the proteins to nitrocellulose membranes. We performed western blots to detect protein as indicated in the figures. We used Secondary antibodies conjugated with peroxidase and revealed with SuperSignal West Pico chemiluminiscent reagent (PIERCE).

6 Loyola et al., 2009 SUPPLEMENTARY LEGENDS TO FIGURES Supplementary Figure 1. Characterization of the HP1α-CAF-1 complex. (A) Coomassie blue stained gel of the HP1α-CAF-1 and.1 complexes as shown in Figure 1A, indicating all the proteins identified by MS. Below the gel is the MS data. (B) Analysis of the CAF-1 complex: cell extract from control cells (mock, same cells but which are not expressing a Flag-tagged protein) or cells stably expressing Flag-p150 (Flag-p150) were incubated with anti-flag agarose beads. Following washes to eliminate the unbound proteins, Flag-p150 was eluted by competition with the FLAG peptide. Proteins present in the Input, Unbound, and Eluate fractions are identified by Western blot. Supplementary Figure 2. Glycerol gradient of the HP1α-CAF-1 complex. (A) Silver staining of a glycerol gradient of the HP1α-CAF-1 complex. The asterisk indicates the fraction were the HP1α-CAF-1 complex appeared (fraction 5). (B) HMTase activity of the glycerol gradient fractions as described in Figure 1C, in which any residue in either of the histones that can be methylated will be labeled. The peak of the -HMTase activity, indicated by the asterisk, co-fractionates with the HP1α-CAF-1 complex (fraction 5). Note that the HMTase with activity towards and H4 migrating in fractions 7-8 corresponds to SKB1. Bottom panel shows the Coomassie-blue stained gel demonstrating equal loads of histones. (C) Comparison of the HMTase activity associated with the HP1α-CAF-1 and.1 complexes, using histone as substrate. Bottom panel shows Coomassie-blue stained gel demonstrating equal loads of histone. 2

7 Loyola et al., 2009 Supplementary Figure 3. K9 monomethylation reduction upon SetDB1 knock-down. We used another sirna against SetDB1 (#2) as a control for possible off target effect in experiments shown in Figure 3C and examined levels of both nuclear and chromatin-associated K9me1. (A) We used increasing amounts of nuclear extract derived from negative control and SetDB1 knockdown cells as indicated for Western Blot. Tubulin showed equal loading. (B) Similarly, we used increasing amounts of nuclear extract (left) and chromatin extract (right) derived from negative control and SetDB1 knock-down cells as indicated and processed them for Western blots. Histone showed equal loading. In addition, for chromatin extract, Ponceau red further confirms equal loading. Supplementary Figure 4. The K9 methylation levels upon SetDB1 knockdown. (A-B) We analyzed increasing amounts of nucleosomal (A) and nonnucleosomal (B) derived from SetDB1 knock-down and control cells. (C) On top, a scheme to illustrate the isolation of HP-1α enriched nucleosomes. We analyzed increasing amounts of the HP1α-enriched nucleosomes by western blot, as indicated. For all the figures, we quantified the bands by densitometry analysis of scanned autoradiography and each of the K9 methylated bands were normalized with their corresponding band to obtain the K9me/ ratio. We compared this number between knock-down SetDB1 and control conditions and calculated the % of reduction taken as 100% the value of control conditions. 3

8 Loyola et al., 2009 SUPPLEMENTARY REFERENCES Dignam, J. D., Lebovitz, R. M., amnd Roeder. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11(5), Groth, A., Ray-Gallet, D., Quivy, J.P., Lukas, J., Bartek, J., and Almouzni, G. (2005). Human Asf1 regulates the flow of S phase histones during replicational stress. Mol Cell 17(2), Marheineke, T. and Krude, T. (1998). Nucleosome assembly activity and intracellular locallization of human CAF-1 changes during the cell division cycle. JBC 273(24),