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

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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

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mock

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BO1

Lalonde et al. Supplementary Figure 5

Lalonde et al. Supplementary Figure 6

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HBO1+BRPF1 w

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MOZ+BRPF1∆(1-

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MOZ+BRPF1 wt

HBO1+BRPF1∆

(1-12

4)

H3

H4

1 2 3 4

Lalonde et al. Supplementary Figure 7