A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster

The incorporation of CENP-A into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to our understanding of CENP-A/Cid loading pathways in Drosophila. Here, we report on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT and/or Caf1/Rbap48. Notably, we identified a novel complex consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. We show that Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition, we demonstrate that CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, our results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones.

2 For protein expression in E. coli, SF-CG2051/Hat1 was amplified by PCR from pMT-SF-CG2051 (see above) and cloned into the NcoI/XhoI digested pET28b vector (New England Biolabs). Similarly, the CENP-A coding region was amplified and cloned into the NdeI/BamHI sites of pET28b to create an in-frame fusion with an N-terminal His-tag. The plasmids were transformed into E. coli BL21(DE3) Rosetta cells.

Extract preparation and affinity purification of CENP-A and CG2051 complexes
Purification from S2 cells: Expression of tagged proteins was induced for 24 h in stably transfected S2 cell lines using 0.5 mM CuSO 4 . All following steps were performed at 4°C. 10 9 cells were harvested, washed with PBS and lysed by addition of 10 ml ice cold Buffer L (10 mM Tris/HCl, pH 8, 340 mM sucrose, 10 mM KCl, 3 mM CaCl 2 , 2 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 0.5 mM benzamidine) to the cell pellet followed by douncing in a glass douncer for 2-3 min. The cell suspension was incubated on ice for 10 min before douncing was repeated. Nuclei were collected by centrifugation at 3700 g for 15 min, mixed with 5 ml Buffer N (30 mM HEPES/KOH, pH 7.9, 150 mM NaCl, 1.5 mM MgCl 2 , 0.05% NP-40, 10% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 1x protease inhibitor cocktail (Roche)), homogenized by douncing for 2-3 min, incubated on ice with gentle stirring for 30 min and finally centrifuged for 30 min at 20,000xg. The resulting nuclear extract was used for affinity purification of tagged CENP-A as well as for western blotting. The remaining chromatin pellet was resuspended in 1 ml Buffer N supplemented with 0.15 U/µl benzonase (Novagen) and incubated for 1 h with gentle stirring before addition of NaCl to 400 mM final concentration and additional incubation for 15 min. The chromatin extract was cleared by centrifugation as before and used for western blot analysis. The supernatant after cell lysis, which contains predominantly cytoplasmic proteins, was cleared again by ultracentrifugation for 1 h at 45,000xg in a Beckmann L-70 ultracentrifuge and used for affinity purification of SF-CENP-A and western blotting. Cell fractionation quality was determined by immunoblotting using the following antibodies against: actin (cytoplasmic marker; 1: 2000; Sigma), transcription factor Zf30C (nuclear extract marker; 1: 20000; home made), histone H3 (chromatin marker; 1: 20000, Millipore).
For purification of EGFP-CENP-A complexes, nuclear extract was precleared with protein Asepharose beads (1:1 slurry) equilibrated in Buffer N before 3 µg of anti-GFP antibody (Novus Biological) per 1 ml extract was added and incubated overnight with rotation. 100 µl of Protein A sepharose slurry were added, incubated for 2 h and centrifuged for 5 min at 2000xg. Beads were extensively washed with Buffer N and finally eluted with 100 mM citric acid (pH 3) for 2 min. Eluted proteins were precipitated with 20% trichloroacetic acid/1 mg/ml deoxycholate, redissolved in 1x Lämmli sample buffer and subjected to SDS-PAGE and Coomassie staining. Gel lanes were cut into 5 equal pieces for further in-gel protease digestion and mass spectrometry (MS) analysis.
Strep-Flag tagged CENP-A or CG2051, respectively, were purified by incubating nuclear extracts with Strep-Tactin Superflow resin (50% slurry in Buffer N; IBA) at ~150 µl slurry per 1 ml extract for 2 h with rotation. Beads were collected by centrifugation at 700xg for 3 min and washed several times with Buffer N. Proteins were eluted twice with 200 µl TBS buffer (30 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) containing 2.5 mM desthiobiotin. Subsequently, eluates were incubated with 100 µl FLAG-M2-agarose (50% slurry in TBS buffer; Sigma) for 2 h with rotation. Beads were collected by centrifugation as above, washed sequentially with TBS/0.1% NP-40, TBS and a.dest. and eventually eluted with 5% acetic acid and 5% acetic acid/10% acetonitrile. For the analysis of posttranslational modifications, CENP-A was purified in an identical manner, except that the starting material was either nuclear extract or cytoplasmic extract and that the final eluates were separated on an SDS-PAGE gel.
Purification from bacteria: Expression of recombinant proteins was induced with 1 mM IPTG for 2 h at 37 °C. Cells were harvested, and lysed by sonication in lysis buffer (10 mM Tris/HCl pH 8, 200 mM NaCl, 0.05% Tween 20, 0.1 mM PMSF, 10 mM imidazole). After centrifugation at 14,000 rpm for 20 min at 4°C, extract containing His-CENP-A was either used directly for Ni 2+ -NTA agarose purification or was combined with bacterial extract containing SF-CG2051 (1:1) and incubated at 25°C for 1 h with gentle agitation followed by incubation with pre-equilibrated Ni 2+ -NTA agarose beads (Qiagen) for 4 h at 4°C. After extensive washing with 20 mM imidazole in lysis buffer, beads were eluted with elution buffer (lysis buffer containing 50 mM NaCl and 300 mM imidazole). One column volume fractions were collected. To control for unspecific binding, SF-CG2051-containing extract was mixed with lysis buffer instead of His-CENP-A extract and purified in the same way.

Quantification of immunoblot signals
To determine the relative distribution of CG2051/Hat1-CENP-A complexes, signal intensities of Hat1 and CENP-A bands were determined after immunoblotting using ImageJ. Taking into account gel loading volume and total extract volume, the absolute amount of protein in a particular cell fraction was calculated and the relative distribution was determined. To calculate the proportion of Hat1 that is associated with CENP-A in each cellular fraction, total amount of protein (arbitrary units) was calculated from signal intensity of the input lane and total volume of input. The signal intensity in the beads lane was then used to calculate the proportion of total protein that was precipitated with CENP-A.

Mass spectrometry and data processing
To identify interaction partners of CENP-A or CG2051/Hat1, the respective proteins were purified by affinity chromatography and elutions were concentrated by speed vac evaporation. Samples were redissolved in 65 µl 100 mM NH 4 HCO 3 /3.8 mM DTT, pH 8.0 and incubated at 56°C for 30 min. 25 µl of 55 mM iodoacetamide (IAA)/100 mM NH 4 HCO 3 was added and incubated at room temperature in the dark before the sample was digested with 1 µg trypsin (Sigma Aldrich) at 37°C for 6 hours.
The gradient profile was as follows: 0-2 min, 4% B; 2-117 min, 4-50% B; 117-122 min, 50-100% B, and 122-27 min, 100% B. The flow rate was 300 nl/min. Samples were analysed using an UltiMate 3000 nano-HPLC system (Thermo Scientific) coupled to a LTQ Orbitrap XL mass spectrometer (Thermo Scientific). The instrument was operated in the data dependent mode selecting the top 10 most abundant isotope patterns with charge 2+ and 3+ from the survey scan with an isolation window of 2 mass-to-charge ratio (m/z). Survey full scan MS spectra were acquired form 300 to 1800 m/z at a resolution of 60,000 with a maximum injection time (IT) of 20 ms, and automatic gain control (AGC) target 1e6. The selected isotope patterns were fragmented by collisional induced dissociation (CID) with normalized collision energy of 35 and a maximum injection time of 55 ms. Data analysis was performed using Proteome Discoverer 1.3 (Thermo Scientific) with search engine Sequest. The raw files were searched against the Drosophila melanogaster BDGP5.68 pep.all database (21,076 entries).
Precursor and fragment mass tolerance was set to 10 ppm and 0.8 Da, respectively, and up to two missed cleavages were allowed. Carbamidomethylation of cysteine and oxidation on methionine were set as variable modifictions. Peptide identifications were filtered at 1% false discovery rate.
For analysis of posttranslational modifications, CENP-A protein bands were excised from an SDS-gel, digested with trypsin or LysC as previously described (Sobieszek et al. 2006) and analyzed using an UltiMate 3000 nano-HPLC system (Thermo Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Scientific) equipped with a Nanospray Flex ionization source. The peptides were separated on a homemade fritless fused-silica microcapillary column (75 µm i.d. x 280 µm o.d. x 10 cm length) packed with 3 µm reversed-phase C18 material (Reprosil). Solvents for HPLC were 0.1% formic acid (solvent A) and 0.1% formic acid in 85% acetonitrile (solvent B). The gradient profile was as follows: 0-2 min, 4% B; 2-55 min, 4-50% B; 55-60 min, 50-100% B, and 60-65 min, 100 % B. The flow rate was 250 nl/min. The Q Exactive Plus mass spectrometer was operated in the data dependent mode selecting the top 12 most abundant isotope patterns with charge >1 from the survey scan with an isolation window of 1.6 mass-to-charge ratio (m/z). Survey full scan MS spectra were acquired from 300 to 1750 m/z at a resolution of 70,000 with a maximum injection time (IT) of 120 ms, and automatic gain control (AGC) target 1e6. The selected isotope patterns were fragmented by higher-energy collisional dissociation (HCD) with normalized collision energy of 25 at a resolution of 17,000 with a maximum IT of 120 ms, and AGC target 5e5. Data analysis was performed using Proteome Discoverer 1.4.1.14 (Thermo Scientific) with search engine Sequest. The raw files were searched against the Drosophila melanogaster BDGP5.68 pep.all database. Precursor and fragment mass tolerance was set to 10 ppm and 0.02 Da, respectively, and up to two missed cleavages were allowed. Carbamidomethylation of cysteine was set as static modification, oxidation of methionine, 6 phosphorylation of serine, threonine and tyrosine, methylation and dimethylation of lysine and arginine, trimethylation of arginine and acetylation of lysine were set as variable modifications.
Peptide identifications were filtered at 1% false discovery rate.

Quench-chase-pulse labeling of newly synthesized CENP-A
To quench SNAP tag activity of existing SNAP-CENP-A, old growth medium was removed, and S2 cells were incubated with 0.9 µM SNAP-Cell Block (NEB) in fresh S2 medium for 30 min at 25°C. times for 10 min each with fresh medium before fixation in 5% paraformaldehyde/0.3% Triton-X100 in PBS. After additional washing in PBS, nuclei were counterstained with DAPI and cells were mounted in vectashield for confocal fluorescence microscopy on a Leica TCS SP5 instrument. Image aquisition was performed using a 63x oil objective with a pixel size of 48.1 nm and by collecting 0.13 µm z-sections spanning the entire nuclei. 3D images were reconstructed and analysed by Imaris v5.1.
Using the new Arena function of Imaris we have generated an "assay" grouping all images into either control or Hat1 group. One image of the Hat1 group was selected and spots were created on that image.
The obtained parameters (including intensity and quality thresholds) for Spot creation were saved ("save for batch") and thereafter applied in the same way to both control and Hat1 images to ensure absolutely consistent image processing. The analyses were performed with and without prior deconvolution using Huygens software. Since the results were virtually the same, only the data without deconvolution are shown. A total of 235-638 centromeres per experiment and treatment condition was analysed in this manner. For comparison of Hat1 RNAi and control samples, mean 7 TMR ± SEM intensities were calculated and statistical significance was determined by two-tailed ttest and Mann-Whitney-test using Prism5.0 software.

Supplementary Figures
Supplementary Figure S1. Multiple sequence alignment of Drosophila CG2051 with Hat1 proteins from other species. Amino acids conserved in at least two species including Drosophila are highlighted in red; amino acids conserved in at least two species but not in Drosophila are denoted in blue and green, respectively. Figure S3. Flag-tagged Drosophila Hat1 and V5-tagged Drosophila CENP-A were overexpressed in Sf9 cells using the baculovirus system and subsequently purified by Flag-affinity chromatography. Aliquots of elutions E1-E4 were subjected to SDS PAGE and Coommassie staining or western blot with anti-Caf1 antibodies. Thereby, co-purification of endogenous (i.e. Sf9 cell) Caf1 with Hat1-CENP-A complexes was detected.