Kwon et al. - 8 -

Supplemental data Kwon et al. “Heterochromatin Protein 1 (HP1) connects the FACT histone chaperone complex to the phosphorylated CTD of RNA polymerase II”

Supplemental experimental procedures

Fly stocks

Su(var)2-5 (FBgn0003607, CG8409) and HP1c (FBgn0039019, CG6990). The HP1c P-element insertion mutant, HP1cf04929, was obtained from Bloomington Stock Center at Indiana University (stock number 18819, FBal0161365). Fly stocks Su(var)2-504/Cyo-GFP (FBal0016506) and Su(var)2-505/Cyo-GFP (FBal0016507) were provided by Sarah Elgin (Washington University, St. Louis, MO).

Antibodies

Anti-FLAG-HRP antibody (A8592), anti-FLAG M2-agarose (F2426), anti-HA agarose (A2095) were purchased from Sigma. Anti-HA rat monoclonal antibody (3F10) (1867423) was from Roche. Anti-H3K36me3 (ab9050), anti-H3K9me3 (ab8898), anti-H3K9me2 (ab1220), anti-H3K9me1 (ab9045), anti-H3K4me2 (ab7766) and anti-histone H3 (ab1791) antibodies were from Abcam. Anti-H3K27me3 antibody (39155) was from active motif. Anti-dSSRP1 (NIG-L1-SHA1) and anti-Spt16 (NIG-L1-SHA2) antibodies were from Cosmo. Anti-RNAPII 8WG16, Anti-RNAPII H14, Anti-RNAPII H5, Anti-RNAP II CTD (4H8) monoclonal antibodies were from Covance. Anti-HP1a antibody (C1A9) was from Developmental Studies Hybridoma Bank (DSHB). Polyclonal antibody against HP1c was raised in rat with peptide antigens as described previously (Brower-Toland B et al., 2007)

Cell lines, extract preparation and protein purification.

Drosophila melanogaster S2 cells were stably co-transfected with D. melanogaster metallothionein (MT) promoter–containing pRmHa3-HP1-C-HA2FLAG2 vectors and pCoBlast (Invitrogen) as described previously (Guelman et al. 2006) and induced with 0.1 mM CuSO4 prior to preparation of nuclear extracts for immunoprecipitation. FLAG-affinity purifications were performed as previously described (Guelman et al. 2006). Cells were collected and washed with ice-cold PBS, before lysed with the lysis buffer containing 10 mM HEPES (pH7.9), 1.5 mM MgCl2, 10 mM KCl, 1% NP-40, 1 mM DTT, 1 mM PMSF and Complete protease inhibitor cocktail tablet (Roche). Nnuclei were pelleted by centrifugation at 5,000 rpm for 5 min at 4°C and extracted using a buffer containing 20 mM HEPES (pH7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM DTT, 1 mM PMSF and Complete protease inhibitor cocktail tablet (Roche). Nuclear extracts were centrifuged at 14,000 rpm for 15 min at 4 °C and then ultracentrafuged at 45,000 rpm for 1.5 hours at 4°C. The NaCl concentration of the extract was subsequently adjusted to 300 mM. Nuclear extracts were incubated with anti-FLAG (M2) agarose beads (Sigma) overnight at 4°C. The beads were washed three times for 10 min in washing buffer (10 mM HEPES (pH 7.9), 300 mM NaCl, 10 mM KCl, 1.5 mM MgCl2, 0.2% Triton X-100, 1 mM PMSF). The complexes were eluted from the beads with elution buffer (0.5 mg/ml triple FLAG peptide in 10mM HEPES-NaOH (pH 7.9), 100 mM NaCl, 1.5 mM MgCl2, 0.05% Triton X-100 and protease inhibitor). For FLAG-HA purification, the elution of the FLAG purification was incubated with HA agarose beads (Sigma), and the complexes were eluted by 500 μg/ml of double HA peptides. A sample of the affinity-purified complex elution was separated on 12% SDS-PAGE and the gel was stained with silver staining. Another sample of the same elution was analyzed by MudPIT analysis as previously described (Lee et al. 2005).

Co-immunoprecipitation assays

For co-immunoprecipitation experiments, and affinity purifications of dHP1 using anti-FLAG or anti-HA antibodies were performed as described previously (Guelman et al, 2006a). S2 cells were transfected using FuGene 6 (Roche) and induced with 0.5 mM CuSO4 prior to preparation of whole-cell extracts for immunoprecipitation. The following antibodies were used in western blots: dHP1a (Generally, in lanes labeled ‘input’ 1/10 of the material used for the binding reaction was loaded.

Expression and purification of recombinant proteins in Sf21 insect cells

cDNAs of dSSRP1, dSpt16, HP1a, HP1b, HP1c and derivatives were subcloned into vector pBacPAK8 carrying a N-terminal FLAG or HA tag. Recombinant baculoviruses were generated and manipulated according to manufacture suggestion (BacPAK expression system (Clontech)). Sf21 insect cells were cultured at 27 °C in the Sf-900 II SFM (Invitrogen) supplemented with 10% FBS (SAFC biosciences), and penicillin-streptomycin (Invitrogen). 48 hours after infection, cells were collected and washed with ice-cold PBS, before lysed in 20 ml of ice-cold lysis buffer (50mM HEPES (pH 7.9), 500 mM NaCl, 2 mM MgCl2, 0.2% Triton X-100, 10% (v/v) glycerol, 0.5 mM EDTA and protease inhibitors). Cell lysates were clarified by ultracentrifugation at 40,000 rpm for 30 min at 4°C, and were subsequently incubated with anti-FLAG (M2), or anti-HA-agarose beads overnight at 4°C. The beads were washed three times with lysis buffer, and bound proteins were eluted twice with 1 column volume of elution buffer (0.5 μg/ml triple FLAG or HA peptide in 50mM HEPES (pH 7.9), 100 mM NaCl, 2 mM MgCl2, 0.02% NP-40 and 10% (v/v) glycerol).

ChIP and quantitative PCR

S2 cells were crosslinked by addition of 10% volume of Paro fixation (50 mM HEPES pH 8.0, 1 mM EDTA, 100 mM NaCl) for 10 min, under non-heat shock conditions or following heat shock at 36.5ºC for 20 min. The cross-linking reaction was quenched with addition of glycine to 125 mM final. The cell pellets were washed by Paro I buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA, 10 mM EGTA, 0.25% Triton X-100) followed by Paro II buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.2 mM NaCl) and then pellets were resuspended by lysis buffer (50 mM HEPES pH 7.5 (KOH), 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% DOC, 0.1% SDS with proteasome inhibitor (Roche)). The lysates was sonicated and the supernatant/DNA was immuniprecipitated with antibodies using Dynabeads (Invitrogen). The beads were washed with lysis buffer followed by DOC (10 mM Tirs-HCl pH 8.0, 0.25 mM LiCl, 1 mM EDTA, 0.5% NP-40 0.5% DOC) and TE, and then beads bound DNA was eluted by Elution buffer (1% SDS, 0.1M NaHCO3). The elution/ Input DNA was incubated with 0.2 mg/ml RNaase A for 30 min 37 ºC, and the crossliking was reversed by 5 mg/ml Proteinase K followed by incubation at 55ºC for 2.5 hr and at 65ºC overnight. DNA was ethanol-precipitated after phenol/chloroform extraction. The DNA pellets were resuspended by 10 mM Tris-HCl pH 8.0. The immuniprecipitated DNA was measure by quantitative Real-Time PCR (qPCR). Each immunoprecipitation was performed and assayed at least six times from at least three separate protein cross-linked DNA extracts for each time point. The percent input shown for each point has a no antibody control value subtracted from the sample value to account for general background. Drosophila Hsp70 primers were previously described (Boehm et al. 2003).

Primers for qRT-PCR

Primers were designed that would amplify transcripts from all six Hsp70 genes, and primers specific to the gene encoding the ribosomal non-heat-shock-sensitive protein Rp49 [Ribosomal protein L32 (RpL32) – FlyBase] were used for normalization.

Corresponding primers: rp49, 5’-AACGTTTACAAATGTGTATTCCGACC-3’ and 5’-ATGACCATCCGCCCAGCATACAGG-3’; Hsp70, 5’-TCATCACAGTTCCAGC -CTACTTCAAC-3’ and 5’-CTGGGTTGATGGATAGGTTGAGGTTC-3’; Hsp83, 5’-CGATTAAGCGACCAGTCGAA-3’ and 5’-CATTCAAGAGCAGTTGTCGTTT-3’; Hsp26, 5’-GTCGAGGACAAGTCCAAGGA-3’ and 5’-GTGGACGACTCCATCTTGGT-3’.

Supplemental References

Guelman, S., Suganuma, T., Florens, L., Swanson, S.K., Kiesecker, C.L., Kusch, T., Anderson, S., Yates, J.R., 3rd, Washburn, M.P., Abmayr, S.M. et al. 2006. Host cell factor and an uncharacterized SANT domain protein are stable components of ATAC, a novel dAda2A/dGcn5-containing histone acetyltransferase complex in Drosophila. Mol Cell Biol 26(3): 871-882.

Lee, K.K., Florens, L., Swanson, S.K., Washburn, M.P., and Workman, J.L. 2005. The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex. Mol Cell Biol 25(3): 1173-1182.

Boehm, A.K., Saunders, A., Werner, J., and Lis, J.T. 2003. Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol Cell Biol 23(21): 7628-7637.

Brower-Toland, B., Findley, S.D., Jiang, L., Liu, L., Yin, H., Dus, M., Zhou, P., Elgin, S.C., and Lin, H. 2007. Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev 21(18): 2300-2311.


Supplemental Figure Legends

Figure S1. MudPIT results of purified samples

(A) MudPIT analysis of Drosophila HP1c. Associated proteins purified from HAFLAG-tagged HP1c, dSSRP1 or dSp16 expressing stable cells under 0.1 mM CuSO4 induction. The table lists the number of nonredundant spectra (total peptides) and the amino acid sequence coverage (SC). Gray indicates the shared interacting proteins of the three HP1 isoforms among the top 32 candidates. (B) Peptides from the three isoforms of HP1 detected by MudPIT in affinity purifications of dSSRP1 or dSpt16.

Figure S2. The interaction between HP1a and dSSRP1 is mediated by the CSD of HP1a

(A)  Schematic representation of HP1a truncation mutants and dSSRP1 and two critical residues of HP1a that are predicted to disrupt either its dimerization (I191E) or its target-binding interface (W200A). Full-length HP1a (FL) contains a chromo domain (CD), a hinge domain (Hin) and a chromoshadow domain (CSD). dSSRP1 possesses the SSRC motif and HMG box domain. The acidic and basic amino acid enriched regions of FACT are indicated in red and blue respectively. (B) HP1a CSD but not CD binds to dSSRP1. Indicated deletion derivatives of GST-HP1a were examined for binding to recombinant HA-dSSRP1. (C) HP1a mutants I191E and W1290A fail to interact with dSSRP1. Indicated HP1a mutants were analyzed for binding to GST-SSRP1. The entire immunoprecipitated material and input (2%) were analyzed by western blot using indicated antibodies in (B-C).

Figure S3. The Alignment of Drosophila HP1s and mouse HP1ß

Sequence alignment of the chromoshadow domains from mouse HP1ß (103-185), and Drosophila HP1a (132-206), HP1b (83-165), and HP1c (63-151) proteins. Amino acid sequences were aligned using CLUSTALX and were presented by PSC geneDoc.

Figure S4. The N-terminal part of dSSRP1 interacts with dSpt16.

(A) Schematic representation of dSSRP1 truncation mutants and dSpt16 (B) GST pull-downs map necessary interaction domains within dSSRP1. Immobilized GST-fusion proteins were incubated with full length of FLAG-dSpt16. Input lane contains 2% of the proteins.

Figure S5. HP1 competes for the binding to dSSRP1 and dSSRP1 inhibits the heterodimerization of HP1

(A-D) In vitro competitive assay using recombinant HP1s with addition of dSSRP1. The molar ratio of HP1a, HP1b, or HP1c and dSSRP1 (A-C) or HP1c and dSSRP1 (D) is 1:0.5 1:1 and 1:2 in lane 4, 5 and 6. Recombinant dSSRP1, HP1a, HP1b and HP1c were mixed with 0.1 mg/ml BSA and immunoprecipitated using anti-HA agarose or glutathione beads. The entire immunoprecipitated material and 2% of input (E) were analyzed by western blot using the indicated antibodies.

Figure S6. HP1a is not enriched in the Hsp70 upon heat shock

Non heat shocked and heat shocked S2 cells were used in chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays with HP1a antibody Numbers below each bar represents the position of real-time PCR primers relative to the Hsp70 transcription start site, as depicted at the bottom of panel E. The intergenic background primer pair targets a region 32 kb downsteam of the last Hsp70 gene at the 87C genomic loci.

Figure S7. HP1c binds H3K9me2/3 but not H3K4me2 and H3K36me3 peptides in vitro

In vitro histone peptide pull-down assay of recombinant HP1 using differentially modified histone H3 peptides. Biotinylated histone H3 peptides (0.5 µg) were adsorbed to streptavidin-coated magnetic beads (Dynal M280). About 0.5 µg of recombinant FLAG-HP1a or FLAG-HP1c was incubated either with magnetic beads alone (Mock) or beads with immobilized peptides, as indicated, for 1h at 4ºC. Unbound faction (Sup), bound faction and 2% of input were subjected to 12% SDS-PAGE gel.

Figure S8. dSpt16 does not bind any form of Pol II CTD peptides in vitro

In vitro CTD peptide pull-down assay of recombinant dSpt16 using differentially phosphorylated CTD peptides. Biotinylated CTD peptides (0.5 µg) were adsorbed to streptavidin-coated magnetic beads (Dynal M280). About 0.5 µg of FLAG-dSpt16 was incubated either with magnetic beads alone (Mock) or beads with immobilized peptides, as indicated, for 1h at 4ºC. Unbound faction (Sup), bound faction and input (2%) were subjected to 10% SDS-PAGE gel.