Supplementary Figure 5. (a) KAT6B especifically acetylates lysine 23 of histone H3 in vitro. Recombinant GST-Flag-tagged KAT6B HAT domains (wild type, and K815L mutant) were assayed for the presence of histone acetyltransferase activity (left-top). Core histones used as substrates were stained with Comassie blue (left-midle). Flag-tagged wild-type and catalytically inactive HAT domains were analyzed in western blot (left-bottom). Recombinant GST-Flag-tagged KAT6B HAT were assayed in western blot for the presence of specific histone H3K23 acetyltransferase activity (right-top). Histone H3 from core histones used as substrates was analyzed in western blot (righ-middle). Flag-tag HAT domains were analyzed in western blot (right-bottom). (b) Recombinant GST-Flag-tagged KAT6B HAT domains were assayed for the presence of histone acetyltransferase activity on recombinant histone H3 (top). Recombinant histone H3 was stained with Comassie blue (midle) and GST-Flag-tagged KAT6B HAT domains were analyzed in western blots (bottom). (c) The expression of HDAC1, 2, 3 and 6 was diminished using specific shRNAs targeting each protein in NCI-N417 cells. Western blot analysis of each HDAC and the acetylation of H3K23 revealed that HDAC1 is involved in H3K23 deacetylation. (d) Quantitative reverse-transcription PCR results confirmed the microarray expression data: twenty one genes (in addition to the four genes shown in Fig. 3f) presented significantly less expression in KAT6B shRNA-depleted cells than in scramble cells. Significance of permutation tests: *, p <0.05; **, p < 0.01; ***, p <0.001.
a b
H3K23ac
Anti-Flag
KAT
6B-H
AT
Total H3
KAT
6B- H
AT-
K81
5LCoomassie
blue
[acetyl-1-14C]-coenzyme A
Anti-Flag
KAT
6B-H
AT
KAT
6B- H
AT-
K81
5L
Coomassie blue
KAT
6B-H
AT
KAT
6B- H
AT-
K81
5L
[acetyl-1-14C]-coenzyme A
Anti-Flag
NC
I-N41
7 sh
RN
A_3
H3K23ac
Lamin B1
HDAC3
NC
I-N41
7 S
cram
ble
NC
I-N41
7 sh
RN
A_2
a
NC
I-N41
7 sh
RN
A_2
b
H3K23ac
Lamin B1
HDAC2
NC
I-N41
7 S
cram
ble
H3K23ac
Lamin B1
HDAC6N
CI-N
417
shR
NA
_6
NC
I-N41
7 S
cram
ble
NC
I-N41
7 sh
RN
A_1
a
NC
I-N41
7 sh
RN
A_1
b
H3K23ac
Lamin B1
HDAC1
NC
I-N41
7 S
cram
blec
d
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
PIAS4
*
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
JHDM1D
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
BNIP3L
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
1.4
0.8
N417 Scramble
N417 shRNA
ADAMTS15
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
ITPRIP
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
N417 Scamble
N417 shRNA
CPS1
0.2
0.4
0.6
0.0
1.0
1.2
1.4
0.8
N417 Scramble
N417 shRNA
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
CNIH3***
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
CDKL4**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
PPAPDC3
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
ICK**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
CACNA1B
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
CMA1
***
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
1.4
0.8
N417 Scramble
N417 shRNA
MIAT
*
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
MRPS10
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
YRDC
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
SLC38A10
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
PVRL2***
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
SEC61A2**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
ARL8A
**
0,0
0,2
0,4
0,6
0,8
1,0
1,2
N417 Scamble
N417 shRNA
0.2
0.4
0.6
0.0
1.0
1.2
0.8
N417 Scramble
N417 shRNA
GNAS-AS1***
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
N417 Scamble
N417 shRNA
ADAMTS4
0.20.40.6
0.0
1.0
1.41.6
0.8
1.2
N417 Scramble
N417 shRNA
**
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
