Extended Data Fig. 3 | H2A.Z enhances the binding of SUV420H1 to promote
its enzymatic activity. a, Liquid scintillation results from analysis of SUV420H1
histone methyltransferase activity, using H2A or H2A.Z mononucleosomes as
substrates (n = 3 biological replicates). b, Mass-spectrometry analysis of
H4K20me2 modification by SUV420H1, using H2A or H2A.Z
mononucleosomes as substrates. c, Western blot analysis of products from
histone methyltransferase assay of SUV420H1 using H2A or H2A.Z
mononucleosomes as substrates. IB, immunoblot. d, Left, mass-spectrometry
analysis of monomethylated and unmethylated H4 histones from chemical
methylation reactions in vitro. Right, western blot analysis and^3 H autography
show that H2A.Z promotes the activity of SUV420H1 on an H4KC20me1
substrate. e, Upper panel, diagram showing four chimaeric mutants of H2A.Z,
with the regions in red replaced with the corresponding regions of H2A.
The sequences of the region containing D97 and S98 of H2A.Z and the
corresponding region of H2A are shown below the diagram (in single-letter
code). Lower panel,^3 H autograph and liquid scintillation analysis of the
methyltransferase activity of SUV420H1 on mononucleosomes containing
H2A.Z chimaeric mutants. f, Western blot analysis following the pulldown of
biotinylated mononucleosomes shows an interaction between SUV420H1 and
mononucleosomes containing wild-type H2A.Z or the H2A.ZD97N/S98K mutant.
g, The interaction between SUV420H1 and H2A, H2A.Z or H2A.ZD97N/S98K was
analysed by LacO/LacI targeting. Scale bar, 5 μm. h, Statistical results from the
LacO/LacI targeting assay of panel g, showing the percentage of cells in which
EGFP–SUV420H1 co-localizes with the indicated histones. Data in panel a are
means ± s.d.; n = 3 biological replicates. Data in panel b are means; n = 2
biological replicates, with dot plot overlaid. Data in panels e, h are
means ± s.e.m.; n = 3 biological replicates; two-tailed unpaired t-test.
The western blots in panels c, d, f, the^3 H autography in panels d, e, the
mass spectrometry in panel d and the fluorescence imaging in panel g
were independently repeated three times with similar results. H4 was
used as a loading control and sample processing control in panels c–f.
For gel source data, see Supplementary Fig. 1. For imaging source data,
see Supplementary Fig. 2.