Supplementary Figure 5. (a) KAT6B especifically acetylates lysine 23 of histone H3 in vitro....
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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 KAT6B-HAT Total H3 KAT6B- HAT-K815L Coomassie blue [acetyl-1- 14 C]- coenzyme A Anti-Flag KAT6B-HAT KAT6B- HA T -K815L Coomassie blue KAT6B-HAT KAT6B- HA T - K815L [acetyl-1- 14 C]- coenzyme A Anti-Flag NCI-N417 shRNA_3 H3K23ac Lamin B1 HDAC3 NCI-N417 S c ramble N CI - N 4 17 shRN A_2a NCI-N417 s h RNA_2b H3K23ac Lamin B1 HDAC2 NC I-N417 Scramble H3K23ac Lamin B1 HDAC6 N CI-N417 shRNA_6 NCI-N417 S c ramble NCI-N417 shRNA_1a NCI-N417 shRN A_1b H3K23ac Lamin B1 HDAC1 NCI-N417 S c ramble c d 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA PIAS4 * 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA JHDM1D ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA BNIP3L ** 0.2 0.4 0.6 0.0 1.0 1.2 1.4 0.8 N417 Scramble N417 shRNA ADAMTS15 ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA ITPRIP ** CPS1 0.2 0.4 0.6 0.0 1.0 1.2 1.4 0.8 N417 Scramble N417 shRNA ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA CNIH3 *** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA CDKL4 ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA PPAPDC3 ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA ICK ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA CACNA1B ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA CMA1 *** 0.2 0.4 0.6 0.0 1.0 1.2 1.4 0.8 N417 Scramble N417 shRNA MIAT * 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA MRPS10 ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA YRDC ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA SLC38A10 ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA PVRL2 *** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA SEC61A2 ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA ARL8A ** 0.2 0.4 0.6 0.0 1.0 1.2 0.8 N417 Scramble N417 shRNA GNAS-AS1 *** ADAMTS4 0.2 0.4 0.6 0.0 1.0 1.4 1.6 0.8 1.2 N417 Scramble N417 shRNA ** Relative expression Relative expression Relative expression Relative expression Relative expression
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Transcript of Supplementary Figure 5. (a) KAT6B especifically acetylates lysine 23 of histone H3 in vitro....
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
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pres
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Rel
ativ
e ex
pres
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Rel
ativ
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sion
Rel
ativ
e ex
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sion
Rel
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