erence cDNA. For immunoprecipitations, transient transfection of plasmid DNA was performed in 293T cells using Polyethylenimine. At 40 h post-transfection, cells were harvested and lysed in 
IP buffer. Cleared lysate was added to Anti-HA Affinity Matrix. Precipitated proteins were separated on 10% SDSPAGE and blotted onto PVDF membranes. The membrane was developed with the appropriate antibodies and ECL. Peptide-binding assays and H3 peptide affinity measurements H3 peptides PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828691 immobilized on streptavidin-conjugated dynabeads were incubated with HeLa nuclear extracts ), extracts from transfected U2OS cells overexpressing wild type or deletion constructs of TAF3 or crude bacterial lysates expressing GSTTAF3-PHD, GST-ING2-PHD, GST-BPTF-PHD, GST-ING4-PHD or GSTBHC-80 in binding buffer for 2 h at 41C. Following extensive washing, bound proteins were eluted and analysed by SDSPAGE and Coomassie brilliant blue R-250 and for experiments with GST-BHC80 and nuclear extracts or transfected lysates by subsequent immunoblotting. Peptide affinity measurements were performed by tryptophan fluorescence essentially as described in Vermeulen et al. Chromatin fractionation Haspin expression in HeLa EGFP-Haspin cells was induced with 1 mg/ml doxycycline treatment for 24 h and uninduced cells were left untreated. Before collecting by centrifugation, the cells were subjected to 10-min incubation with 1 mM Okadaic acid to maintain phosphorylation. Cell pellets were resuspended in hypotonic lysis buffer vanadate, 5 mM sodium pyrophosphate and 1 mM okadaic acid) and lysed using 27G syringe. One volume of 2 extraction buffer was added dropwise and lysates were tumbled for 30 min at 41C followed by centrifugation for 45 min at 225 000 g and 41C. The supernatant was set aside as the soluble extract, and the pellet was washed twice in wash buffer by homogenizing and centrifugation at 41C for 30 min at 225 000 g, and the resulting supernatants were collected as wash 1 and wash 2, respectively. The chromatin pellet was homogenized in a nucleosome isolation buffer, and the samples were incubated at 371C for 5 min, prior to incubation with 10 U/ml micrococcal nuclease at 371C. The reaction was stopped by the addition of 10 mM EGTA and cooling on ice. The soluble chromatin fraction was obtained by centrifugation at 10 000 g for 10 min at 41C. siRNA knockdown, immunofluorescence and live-cell imaging U2OS-GFP-TAF5 cells were grown on coverslips and transfected with either siRNA oligos only or siRNA oligos along with H2B-dsRed DNA by using the Dharmafect 1 reagent or Dharmafect Duo, respectively. Haspin siRNAs, nontargeting control siRNA and siGlo Red transfection indicator oligos were purchased from Dharmacon. Haspin siRNA knockdown with both of the oligos gave comparable results. For immunolabelling, 40 h after transfection, cells were fixed with 4% formaldehyde/PBS for 20 min, permeabilized using 0.2% Triton X-100, blocked in 5% FBS/PBS for over 30 min; then incubated with anti-GFP, anti-H3T3ph, anti-H3S10ph in 5% FBS/ PBS for 1 h at room temperature followed by a 30-min incubation in goat Neuromedin N site anti-mouse IgG-Alexa488 or goat anti-rabbit IgG-Alexa568. To detect DNA, 10 mg/ml DAPI was used. Fluorescence microscopy was performed using a Zeiss 510 Meta confocal microscope with a 63 1.4NA Plan-ApoChromat objective. For live-cell imaging, transfected cells in LabTekII four-well chamber slides were released from an 18-h thymidine block to obtain as many cells in the same phase of th