Molecular Cell, Volume 24

Supplemental Data

Yng1 PHD Finger Binding to H3 Trimethylated at K4

Promotes NuA3 HAT Activity at K14 of H3 and

Transcription at a Subset of Targeted ORFs Sean D. Taverna, Serge Ilin, Richard S. Rogers, Jason C. Tanny, Heather Lavender, Haitao Li, Lindsey Baker, John Boyle, Lauren P. Blair, Brian T. Chait, Dinshaw J. Patel, John D. Aitchison, Alan J. Tackett, and C. David Allis

Figure S1.

High pressure gel filtration chromatography was performed by using a Bio-Silect SEC

400–5 column (Bio-Rad). Cell lysate (prepared identically to the immunoisolations in

Figure 1A) from ~ 1 X 108 YNG1::TAP-HIS3 cells was loaded onto the column and 250

µL fractions were taken at a flow rate of 1mL/min. The presence of Yng1-TAP was

determined by Western-blotting. A calibration curve was created using Bio-Rad Gel

Filtration Standards.

Figure S2.

Control for H3 sample loading and peptide methylation in Figure 2 pull-down assays.

Figure S3.

(a) 1H15N-HSQC spectra with assignments of the PHD domain free in solution.

(b) 1H15N-HSQC spectra with assignments of the PHD domain bound to the H31-

9K4me3 peptide.

Figure S4.

A histogram of differences in the chemical shift values (∆δ) of the backbone 1H and 15N

resonances of the PHD domain in the free and H31-9K4me3 peptide bound form as a

function of residue number. ∆δ for each residue was calculated from the equation ∆δ =

[(∆δH)2 + (0.1DdN)2]1/2 where DdH and DdN are the chemical shift differences in the 1H

and 15N resonances, respectively.

Figure S5.

Quantitative rtPCR was used to determine differences in transcription levels of Yng1

targeted ORFs. ORFs were selected from the top 50 genomic binding sites of Yng1,

and they covered a range of transcriptional levels. These overall cDNA levels were

normalized to the expression levels of actin in wild-type and W180E mutant strains.

The transcription level was normalized to wild-type as 100%. Error bars show the

standard deviation of triplicate analyses.

Supplemental Experimental Procedures

In Vivo Binding Assay

Extracts were made as described for the Yng1-TAP immunopurification with the

following changes. Frozen, lysed cells (1 g/ pull-down condition) were extracted in 500

mM extraction buffer (500 mM NaCl, 20 mM HEPES pH 7.9, 25% glycerol, 1.5 mM

MgCl2, 0.2 mM EDTA, 1 mM PMSF, Complete Mini EDTA-free [Roche], 0.2% Triton X-

100) for 1 hour at 4°C. Extracts were then diluted to 150 mM NaCl with ‘no-salt’

extraction buffer, mixed with 2.5 µg of biotinylated histone peptide-linked Dynabeads

(M-280 Streptavidin, Dynal) at the ratio of 2.5 µg /100 µL beads and nutated for 30

minutes at 4°C. Peptide-linked Dynabeads and associated proteins were then washed

five times in 300 mM KCl wash buffer (300 mM KCl, 20 mM HEPES pH 7.9, 0.2% Triton

X-100), and one time in a buffer containing 4 mM Hepes pH 7.5 and10 mM NaCl.

Peptide-bound proteins were eluted in boiling SDS-PAGE loading buffer, resolved on

Novex 4-20% gradient gels, and probed with antibodies recognizing the PrA tag (DAKO

P0450). Total represented 0.5% of input. Unmodified, K14 acetylated, K9/14

acetylated, and K4 mono- and trimethylated H3 peptides were from Upstate Biotech

(UBI). Dimethylated K4, trimethylated K9, trimethylated K27, trimethylated K79 H3, and

trimethylated K20 H4 were synthesized at the Proteomics Resource Center of The

Rockefeller University.

Protein Preparation of Yng1 PHD finger

The Yng1 PHD finger constructs were made using pGex6p vector with an N-terminal

glutathione S-transferase (GST) tag. Proteins were over-expressed in the Escherichia

coli host cell Rossetta2 (Novagen) induced overnight by 0.4 mM isopropyl β-D-

thiogalactoside at 15ºC in LB medium supplemented with 0.1 mM ZnCl2. Harvested

cells were disrupted using Emulsiflex-C5 Homogenizer (Avestin) at 4 ºC. After

centrifugation of the cell lysate, the GST-fusion protein was purified by GST affinity

column from the supernatant, followed by tag cleavage using PreScission protease (GE

Healthcare). Finally, the PHD finger was directly purified from the digestion mixture by

size-exclusion chromatography on Superdex 75 pg 26/60 column using an AKTA

Purifier system (GE Healthcare). The construct containing residues 141-219 of full

length Yng1 was used for NMR studies. 15N and 13C,15N-labeled protein samples were

produced by an improved method (Marley, 2001), and were concentrated to ~0.3-0.5

mM in 50 mM KCL, 20 mM Na/K phosphate pH 7.5 and 2mM dithiothreitol prior to use.

For fluorescence polarization assays, a better behaved shorter construct containing

fragment 153-204 of full length protein was used to produce both native and W180E

mutant Yng1 PHD fingers. The W180E mutant was made using the Quick Change site-

directed mutagenesis kit (Stratagene) and was purified as described for the native

protein.

NMR Spectroscopy

NMR spectra were collected at 20ºC on Bruker DRX600 and DRX800 spectrometers

equipped with z-axis gradient triple resonance cryoprobes. Sequence-specific backbone

1HN, 15N, 1Hα, 1Hβ, 13Cα and 13Cβ were determined using HNCACB, CBCA(CO)NH,

HNCA, HN(CO)CA and HN(CO)HAHB (Grzesiek, 1992; Kay, 1994; Muhandiram, 1994).

Secondary structure and side-chain assignments were obtained from HCCH-TOCSY

experiments (Ikura, 1990). 3D 1H-15N/13C NOESY-HSQC (mixing time 120 ms) spectra

provided distance restraints used in the final structure calculations. The assignment of

the non-labeled-peptide H31-9K4me3 was made using a combination of filter

experiments such as TOCSY and NOESY with half-filter in both ω1 and ω2 to select 12C

& 14N spin systems. 2D ω1/ω2-filter 1H-12C NOESY (mixing time 120 ms) spectra

provided intra H31-9K4me3 distance restraints, whereas 2D ω2-filter 1H-12C NOESY and

3D ω2-edit 1H-13C NOESYHSQC (mixing time 120 ms) spectra provided protein-peptide

distance restraints. Backbone dihedral angle restraints (φ and ψ) were predicted using

the program TALOS (Cornilescu, 1999) based on the chemical shifts of HA, N, CA, CB

and CO. Amide protons either protected from exchange or involved in hydrogen bonds

were identified by the presence of NH resonances in 1H15N-HSQC spectra recorded in 2

hours after the addition of 100% 2H2O to a lyophilized, 15N-enriched protein in the

complex with H31-9K4me3 peptide. The hydrogen bond acceptors were identified

according to distinctive neighboring NOEs observed in a pre-folded structure. All

spectra were processes with TOPSPIN 1.0 (Bruker Biospin GmbH) and analyzed with

CARA 1.3 (Keller, 2004).

Structure Calculations

The structure of the YNG1 PHD domain free in solution and in the complex with H31-

9K4me3 peptide were calculated by X-PLOR (Brünger, 1993) using torsion angle

dynamics protocol. NOE distance restraints were derived from NOESY data. The final

structure calculations employed a total of 2701 NOE restraints that comprise 746

intraresidue, 193 sequential, 107 medium-range, and 262 long-range NOEs for the free

form and 741 intraresidue, 193 sequential, 122 medium-range, and 254 long-range

NOEs for the bound form. The restraints used for structural calculations and the

structural statistics are summarized in Table S2. The structural coordinates of YNG1

PHD domain free and in the complex with H31-9K4me3 peptide have been deposited in

the Protein Data Base under accession codes XXX and XXX, respectively.

Supplemental References

Brünger, A. T. (1993). X-PLOR Version 3.1: A System for X-Ray Crystallography and

NMR (New Haven, CT: Yale University Press).

Cornilescu, G., Delaglio, F., and Bax A. (1999). Protein backbone angle restraints from

searching a database for chemical shift and sequence homology. J Biomol NMR 13,

289-302.

Grzesiek, S., and Bax, A. (1992). Improved 3D Triple-Resonance NMR Techniques

Applied to a 31-Kda Protein. Journal of Magnetic Resonanc 96, 432-440.

Ikura, M., Kay, L.E. and Bax, A. (1990). A novel-approach for sequential assignment of

H-1, C-13, and N-15 spectra of larger proteins - heteronuclear triple - resonance 3-

dimential NMR-spectroscopy - application to calmodulin. Biochemistry 29, 4659-4667.

Kay, L. E., Xu, G. Y., and Yamazaki, T. (1994). Enhanced-Sensitivity Triple-Resonance

Spectroscopy with Minimal H2O Saturation. Journal of Magnetic Resonance Series A

109, 129-113.

Keller, R. (2004). The Computer Aided Resonance Assignment, ISBN 3-85600-112-3, CANTINA Verlag.

Muhandiram, D. R., and Kay, L. E. (1994). Gradient-Enhanced Triple-Resonance 3-

Dimensional Nmr Experiments with Improved Sensitivity. Journal of Magnetic

Resonance Series B 103, 203-216.