Supplementary Figure 6: Highly purified Tcf1 catalyzes deacetylation of histone H3K9Ac peptide.

(a) Purification of Tcf1 by FPLC. Shown is the chromatogram elution profile for Tcf1 run over a superdex 75 10/300 GL column. The UV absorbance trace at 280 nm is shown in black and that at 260 nm is shown in gray. Fractions were collected every 0.5 ml starting at 1 ml. The major peak was eluted at 8–10 ml, corresponding to fractions 14-18, which were analyzed by SDS-PAGE and Coomassie Blue staining (see inset). Fraction 15 containing Tcf1 with the highest purity was used in all the HDAC assays with histone proteins or peptide as substrates (Fig. 6 and panels b-d in this figure).

Fraction 15 was immunoblotted with a house-made Tcf1 antiserum, which confirmed the major component in the preparation was Tcf1. The minor amounts of proteins of smaller molecular weights were also reactive to the Tcf1 antiserum, suggesting that these smaller proteins are likely degradation products from purified Tcf1.

(b) Purity of FPLC-purified Tcf1 as determined by silver staining. Fractions 14-17 from an independent FPLC purification was resolved on SDS-PAGE, and silver-stained using the “Pierce Silver Stain for Mass Spectrometry”.

(c) Detection of histone H3 (1-21) in K9 acetylated and deacetylated forms with MALDI. Commercially available histone H3(1-21)K9Ac and H3(1-21) peptides were suspended in an HDAC assay buffer at 100 µM and detected on MALDI at m/z of ~2296 and ~2254, respectively. Each peptide appears as a series of multiple consecutive peaks with an one Dalton spacing and a relative peak height determined by the natural abundance of principally carbon, hydrogen and sulfur isotopes.

(d) Validating the identity of the product in Tcf1-catalyzed HDAC assay using H3(1-21)K9Ac peptide. FPLC-purified Tcf1 was incubated with 100 µM H3(1-21)K9Ac peptide for 2 hrs, and the 2,254 Da product and 2,296 Da remaining substrate peptide were detected with MALDI as in Fig. 6b. The product and substrate were further analyzed with Laser-Induced Fragmentation Time of Flight (LIFT). The product (lower panel) had the identical amino acid sequence as the substrate (upper panel), except that the product sequence displays Lysine 9 in its deacetylated form, with 42 Da (equal to an acetyl group) reduction in mass at this particular residue.

(e) Kinetics calculation of Tcf1-catalyzed deacetylation of H3(1-21)K9Ac peptide. The reciprocal of reaction velocity (1/V) and that of H3(1-21)K9Ac peptide substrate concentration (1/[S]) were plotted using data from Fig. 6d. Linear trendline was added using Excel, and the equation and correlation coefficient (R²) are shown in the figure. Determined from the y-intercept, Vmax = 1/1,401,754 mM/sec = 0.71×10^–6 mM/sec. Determined from the slope, Km = 30,758/1,401,754 mM =0.022 mM.