1 Supporting Online Material : Avdic et al. Structure of the trithorax group protein Ash2L reveals a Forkhead-like DNA binding domain. Sabina Sarvan 1,2,6 , Vanja Avdic 1,2,6 , Véronique Tremblay 1,2,6 , Chandra-Prakash Chaturvedi 3,4 , Pamela Zhang 1,2 , Sylvain Lanouette 1,2 , Alexandre Blais 1,2 , Joseph S. Brunzelle 4 , Marjorie Brand 3,4 and Jean-François Couture 1,2 Nature Structural & Molecular Biology: doi:10.1038/nsmb.2093
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Supporting Online Material : Avdic et al. Structure of the ... · (version 4.1.1, Applied Photophysics Ltd., Leatherhead, Surrey, U.K.). The raw spectrum for each protein sample was
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Supporting Online Material : Avdic et al.
Structure of the trithorax group protein Ash2L reveals a Forkhead-like DNA
Supplementary Figure 1. Ash2L is composed of a C4 zinc finger and an evolutionary conserved HWH domain. A protein sequence alignment of Ash2LN from Homo sapiens (Hs) (numbered accordingly to the isoform 1 of the Ash2L (uniprot.org)) with the corresponding N-terminal domains of Danio rerio (Dr), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce) and Schizosaccharomyces pombe (Sp) Ash2L proteins. Ash2LN β-strands and α-helices are depicted as cylinders and arrows, respectively. Positions with 100-80%, 80-60% and less than 60% of amino acid conservation are represented respectively in dark, medium and pale blue.
Supplementary Figure 2. DNA binding affinity of Ash2LN for HS2. The apparent equilibrium dissociation constant was calculated by increasing Ash2LN concentration (500nM–15000nM). The experimental data are reported as a ratio of Ash2LN-bound DNA (Bound) to fully Ash2LN-bound DNA (BoundMax) against protein concentration (Log10). The apparent equilibrium dissociation constant corresponds to the protein concentration yielding a Bound × BoundMax -1 of 50%. The graph represents an average of four independent titration experiments and error bars represent the s.d..
Supplementary Figure 3. Ash2LN is dispensable for the stimulation of MLL1 methyltransferase activity. Methyltransferase assays were performed as previously described 1 using either MLL1 catalytic domain (a), WDR5-RbBP5-Ash2L-MLL1 (b), WDR5-RbBP5-Ash2LN-MLL1 (c) or WDR5-RbBP5-Ash2LC-MLL1 (d). The methyltransferase assays have been performed in tri-plicate and the error bars represent the s.d.
Supplementary Figure 4. Mutation of Ash2LN does not impair protein folding. Collection of circular dichroism spectra for the wild type and mutant proteins was performed using a Chirascan Circular Dichroism Spectrometer (Applied Photophysics Ltd., Leatherhead, Surrey, U.K.). Each protein (or buffer) sample was placed in a 0.1mm path-length cylindrical cuvette for analysis, and seven replicate scans were obtained. Processing of raw spectra was performed using the Pro-data Suite software package (version 4.1.1, Applied Photophysics Ltd., Leatherhead, Surrey, U.K.). The raw spectrum for each protein sample was corrected for baseline anomalies by subtraction of the spectrum for a corresponding buffer blank, and replicate scans were averaged. These corrected spectra were smoothed using a 3-point window, which showed no discernable systemic variability in the residuals.
Supplementary Figure 5. Role of Ash2LN in the NF-E2-mediated recruitment of MLL2 to β-globin LCR. Schematic representation of Ash2LN role in sensing the binding of NF-E2 to the HS2. The binding of NF-E2 is recognized by Ash2LN, either mediated by a direct interaction between NF-E2 and Ash2L, recruitment of an additional protein or changes in DNA structure upon binding of NF-E2 to HS2 (hypotheses illustrated as ?). The binding of Ash2L to HS2 concomitantly tethers MLL2 along RbBP5 and WDR5 to this loci and allow the methylation of the neighboring histone H3.
Supplementary Figure 6. Ash2L is enriched at promoter regions of genes harboring motif #1 (a) Validation of putative Ash2L target genes using ChIP in Hela cells for motif #1. CHiP assays were performed with an anti-Ash2L antibody and protein A agarose (Zymed). Reverse cross-linked DNA was PCR-amplified and separated on a 3% agarose gel (TAE 1X). (b) Ash2L stimulates luciferase expression. Hela cells were transfected with the indicated firefly luciferase reporter constructs and pCMV-Ash2L. Normalized firefly luciferase activities were divided by the protein concentration and reported as fold of induction over empty plasmid. (c) Ash2L α5-helix is required for induction of luciferase activity. Ash2L wild type or mutants are detected by western blot with indicated antibodies (inset). Luciferase activity in cells transfected with pGL3-promoter and various constructs of Ash2L. Activity is reported as in b. Each experiment was performed a minimum of three times and error bars represent s.d.
Ligand/ion 1 Water 7 B-factors (Å2) Protein 27.4 Ligand/ion 43.6 Water 27.3 R.m.s. deviations Bond lengths (Å) 0.021 Bond angles (°) 1.909 The data set has been collected on a single crystal * Highest resolution shell is shown in parenthesis
1. Avdic, V. et al. FASEB J (2010). 2. Couture, J.F., Collazo, E., Ortiz-Tello, P.A., Brunzelle, J.S. & Trievel, R.C. Nat
Struct Mol Biol 14, 689-95 (2007). 3. Otwinowski, Z. & Minor, W. Processing of X-ray Diffraction Data Collected in
Oscillation Mode, 307-326 (Academic Press, New York, 1997). 4. Zwart, P.H. et al. Methods Mol Biol 426, 419-35 (2008). 5. de La Fortelle, E. & Bricogne, G. Methods in Enzymology 276, 472-494 (1997). 6. Cowtan, K.D. & Main, P. Acta Crystallogr D Biol Crystallogr 49, 148-57 (1993). 7. Abrahams, J.P. & Leslie, A.G. Acta Crystallogr D Biol Crystallogr 52, 30-42
(1996). 8. Perrakis, A., Harkiolaki, M., Wilson, K.S. & Lamzin, V.S. Acta Crystallogr D
Biol Crystallogr 57, 1445-50 (2001). 9. Emsley, P. & Cowtan, K. Acta Crystallogr D Biol Crystallogr 60, 2126-32
(2004). 10. Vagin, A.A. et al. Acta Crystallogr D Biol Crystallogr 60, 2184-95 (2004). 11. Demers, C. et al. Mol Cell 27, 573-84 (2007). 12. Linhart, C., Halperin, Y. & Shamir, R. Genome Res 18, 1180-9 (2008). 13. Ji, H. et al. Nat Biotechnol 26, 1293-300 (2008). 14. Dennis, G., Jr. et al. Genome Biol 4, P3 (2003). 15. Blais, A., van Oevelen, C.J., Margueron, R., Acosta-Alvear, D. & Dynlacht, B.D.