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Supplemental Lalonde et al. Page 1
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Fig. 1. Characterization of the two PHD fingers in the PZP domain of
BRPF1. (a) Peptide pull-down assays with different biotinylated peptides and
recombinant PHD1 finger fused to GST were analyzed by Western blotting with anti-
GST antibody (WB:α-GST). (b) Superimposed 1H,15N HSQC spectra of BRPF1 PHD1,
collected as indicated H3 peptides were titrated in. The spectrum is color-coded
according to the protein-peptide ratio. Addition of H3 (3-10) induced no changes in the
NMR spectra of PHD1, implying that this module does not bind H3 lacking the first two
N-terminal residues and that the Ala1-Arg2 sequence is required for robust interaction. A
lack of any significant resonance perturbations upon titrating H3A1ac indicated that the
PHD1 finger is unable to bind this peptide. (c) Alignment of known H3K4me0-binding
PHD domains. Amino acid conservation is shown as well as important regions for H3 tail
interaction. (d) Peptide pull-down assays with unmodified H3 peptide and two point
mutants of recombinant GST-PHD1 were analyzed by Western blotting with anti-GST
antibody (WB:α-GST). The F292A and D294A mutations decrease the affinity with the
H3 peptide. Quantification is also shown on the right. (e) Superimposed 1H,15N HSQC
spectra of BRPF1 PHD2 (0.1 mM), collected as dNMPs were titrated in. (f)
Superimposed 1H,15N HSQC spectra of BRPF1 PZP, recorded during titration of
indicated H3 peptides.
Supplementary Fig. 2. MYST subunits ChIP-seq signal intensities around the
transcription start site (TSS) of genes in human RKO cells correlates globally with gene
expression levels. ChIP global profiles of (a) H3K4me3 (b) ING5 (c) ING2 and (d)
BRPF1/2 were produced using UCSC genome browser gene definition and the Python
package (see Methods). The binning of genes by their expression levels in RKO cells was
performed by subdividing genes in four equal categories (the fourth quartile includes the
most highly expressed genes).
Supplementary Fig. 3. Control heatmaps for ChIP-seq signals on +/- 5kb surrounding
TSS of genes are shown. Genes were sorted by gene expression level (see Methods) from
high (top) to low (bottom) and the signal were corrected over reads per million (rpm).
Heat Maps of Rabbit IgG negative control (a) or input (b).
Supplemental Lalonde et al. Page 2
Supplementary Fig. 4. BRPF2 antibody recognizes both BRPF2 and BRPF1. (a)
Western blot on IPs (IgG, anti-Flag or anti-BRPF2) performed on chromatin fractions
from K562 cells stably expressing BRPF1-Flag. (b) Western blots with anti-Brpf2 and
anti-Flag on purified BRPF3 and BRPF1 native complexes from K562 cells (stably
expressing BRPF3-Flag or BRPF1-Flag). An empty-Flag K562 cell line was used as a
negative control.
Supplementary Fig. 5. BRPF1/2 interacts with HBO1. 293T cells were transfected with
the flag-HBO1 or the equivalent mock expression plasmids. Flag IPs were performed on
WCE and purified proteins were loaded on SDS-PAGE for detection by WB with
BRPF1/2 antibody.
Supplementary Fig. 6. HBO1-BRPF1 complex acetylates H3K14. HAT assays with the
purified HBO1-BRPF1 complex (with ING5 and hEaf6) were performed on chromatin
and on (a) H3wt(1-21) and H3K14ac or (b) H3wt(21-44), H3K27me3 and H3K26me3
histone peptides and spotted on membranes for liquid scintillation counting. Values are
based on three independent experiments with standard error. Acetylation of H3K14
clearly inhibits global acetylation of the peptide while no significant change was
observed with H3K27me and H3K36me3, arguing for H3K23 acetylation.
Supplementary Fig. 7. Deletion of BRPF1 N-terminus also changes the histone tail
specificity of the associated acetyltransferase. 293T cells were cotransfected with the
indicated expression plasmids, with HA-tagged catalytic subunits and Flag-tagged
scaffold subunits. HA-ING5 and HA-hEaf6 expression plasmids were also cotransfected
for each purification. Flag IPs were performed on WCE and were eluted with 3xFlag
peptides. Purified complexes were used in HAT assays and acetylated histones were
separated by SDS-PAGE and revealed by fluorography. In order to detect changes in
acetylation specificity, in contrast to Fig. 6b, we normalized the assays by using the same
level of total activity (measured by liquid assay) on chromatin rather than correcting with
the activity on free histones.
a
e
Lalonde et al. Supplementary Figure 1
b
c
d
f
a ING5b
c
Lalonde et al. Supplementary Figure 2
dING2 BRPF1/2
H3K4me3
IgG
-4000 +40000bp from TSS
RPM
0 84
gene
s so
rted
by e
xpre
ssio
n le
vels
2000
6000
10000
14000
2000
6000
10000
14000
Input
-4000 +40000bp from TSS
RPM
0 1.40.7
2000
6000
10000
14000
gene
s so
rted
by e
xpre
ssio
n le
vels
2000
6000
10000
14000
Lalonde et al. Supplementary Figure 3
a b
empty-Flag
Brpf3-Flag
Brpf1-Flag
Brpf3-Flag
Brpf1-Flag
anti-Brpf2 anti-Flag
Lalonde et al. Supplementary Figure 4
ba
inputIgG ChIP
Flag ChIP
Brpf2 ChIP
anti-Flag
Brpf1-Flag chromatin
Brpf1-Flag
Brpf2 anti-Brpf2 Purified complexes
Input
Flag-IP
FT
Brp
f1/2
WB
mock
Flag-H
BO1
Lalonde et al. Supplementary Figure 5
Lalonde et al. Supplementary Figure 6
0
5000
10000
15000
20000
25000
30000
35000
HAT
act
ivity
(cpm
)
0
2000
4000
6000
8000
10000
12000
14000
HAT
act
ivity
(cp
m)
a
b
+HA-ING5, HA-hEaf6
HBO1+BRPF1 w
t
MOZ+BRPF1∆(1-
124)
MOZ+BRPF1 wt
HBO1+BRPF1∆
(1-12
4)
H3
H4
1 2 3 4
Lalonde et al. Supplementary Figure 7