Measuring Dynamic Histone Acetylation
in vivo.
Jakob Waterborg
University of MissouriKansas City, U.S.A.
Email:
WaterborgJ@umkc.edu
© 2002 Waterborg - UMKC
Wolffe95
Papers discussed
Saccharomyces cerevisiae, baker’s yeast
Dynamic acetylation:
• Waterborg (2001)
Biochemistry 40, 2599-2605
Histone preparation and fractionation:
• Waterborg (2000)
J. Biol. Chem. 275, 13007-13011
Chlamydomonas reinhardtii algae
Dynamic acetylation:
• Waterborg (1998)
J. Biol. Chem. 273, 27602-27609
Histone preparation and fractionation:
• Waterborg et al. (1995)
Plant Physiol. 109, 393-407
© 2002 Waterborg - UMKC
Function of Histone Acetylation
30 nm
11 nm
HIGH in histone H1
+
charged
histone
tail
acetylated
histone tail
© 2002 Waterborg - UMKC
Often low in histone H1
Histone Acetylation Enzymes
Lysine
in histone:
cell membrane
N
O
C C g
a b
C
C
C
d
O
O
e
C
N+
O
[3H]-Ac
C
O
Acetyl-CoA
HAT (Histone
-
HDAC
AcetylTransferase)
C
O
(Histone
DeACetylase)
CoA
N
Uncharged
Acetyl-Lysine
in histone
C
O
C
C
C
C
e
C
C
N
C
O
© 2002 Waterborg - UMKC
DNA
P
O
Acetylated Histones – a choice
N-terminus of histone H3
++
+ +
H3N- ARTKQTARKS
+
9
5
m
m
+
++
TGGKAPRKQL
18
14
+
++
ASKAARKSAP…
23
27
3-6 Ac
m
H3 : 3-6Ac
H2B : 3-6Ac
H4 : 4 or 5 Ac
N-terminus of histone H4
+
++ ++
+
+ +
+
Ac-HN- SGRGKGGKGL GKGGAKRHRK ILR…
8
16
20
5
12
4 or 5 Ac
(m)
Note the “standard” colors for the core histones
© 2002 Waterborg - UMKC
H2A : 1-2Ac
Preparation of Histones
1. Break cells – collect nuclei
Yeast: glass-beading (fast)
Chlamydomonas: cell-wall-less mutant
2. Extract with acid: 0.4 N H2SO4 or HCl
Chaotropic extraction by
>20% guanidine.HCl pH 6.8
3. Collect histones by precipitation
(acetone or TCA)
Dialysis into 2.5% acetic acid
(remove all salt)
Lyophilization until dry
© 2002 Waterborg - UMKC
Separation of Histones
4. Reversed-phase C18 HPLC
Absorbance
0-60% acetonitrile gradient
H2B
H1.2
H4
H2A
H3
H1.1
20
40
60
Chlamydomonas histone extract
5. Acid-Urea gel electrophoresis
© 2002 Waterborg - UMKC
80 min
SDS or AU Gel Electrophoresis ?
AU (Acetic Acid – Urea)
SDS
Gel system:
1M acid, pH ~3 in 5-8 M urea
size + charge
Separation by: size
electrode:
_
+
++
++
H3
H4
135 aa
135 aa
H3
H3
H4
102 aa
electrode:
+
++
+
H4
102 aa
_
Densitometry of Coomassie dye
© 2002 Waterborg - UMKC
Gel scans: alfalfa histones (Waterborg, 90’s)
How to Detect Histone Acetylation?
(fluorograph)
Alfalfa histone H4
Lysine acetylation at
5, 8, 12, 16 and 20
© 2002 Waterborg - UMKC
Measure Post-translational Acetylation
AcLysines:
5
Fluorograph
4
3
2
1
0
Chlamydomonas H4
60 min *Ac label
Coomassie
Fluorograph
top of gel +
© 2002 Waterborg - UMKC
2 min *Ac pulse label
+chx
– top of gel
Acetylation of Yeast histone H4
30 min
120 min 120 min
120 min
[3H]-Ac
[3H]-Ac
[3H]-lysine
+
+
[3H]-Ac
–
–
cycloheximide
Ac
Ac
Coomassie
Fluorograph (film)
© 2002 Waterborg - UMKC
Acetylation of histone H3
Chlamydomonas H3
Saccharomyces H3
60min *Ac
Coomassie
Coomassie
2 min *Ac
+cycloheximide
30 min *Ac
+cycloheximide
54 3 21 0
3
5
4
0
3
4
2
5
1
(top) AUT gel
© 2002 Waterborg - UMKC
(bottom)
(top) AUT gel
2
1
0
(bottom)
Acetate Labeling in Yeast
Outside yeast cell
Inside yeast cell
(glucose in YPD growth medium)
Glucose represses
Active acetate transport
pH7 pH4
pH7
*Ac added: 2,000 nM
H+
Ac–
1,980 nM
<600 nM
Diffusion
HAc
20nM
1%
> 1,400 nM
slow equilibration
to 2,000 nM
HAc
H+
rapid accumulation Ac–
to >> 2,000 nM Ac
HAc pKa = 4.76
Nelson (1982) J.Biol.Chem. 257, 1565
© 2002 Waterborg - UMKC
*Acetate Labeling in Chlamydomonas
Coom 0
2
6
12
20 60 min
Coom 0
2
6 12 20 60 min
5
5
0
H3
100
2
50
T1/2
0
5
3
4
0
max
0
1 Ac
label
1
50
H4
100
2-5 Ac
label
0
10
© 2002 Waterborg - UMKC
20
min 60
0
10
20
min 60
Pulse-chase Label Protocol
exponential decay of specific label
% specific label
1
50
"pulse label"
Specific radioactivity
100
0.1
half-life
T1/2
0.01
0
15time 20
10
"chase"
0
0
5
10
20
30
40
50
time
© 2002 Waterborg - UMKC
60
70
80
90 100
Specific radioactivity (%)
Pulse-chase *Ac Labeling
100
Pulse = 2 min 10M *Acetate.
Chase = at 2 min: add 1 volume of
growth medium with 9.34 mM
NH4Ac (1,000x excess).
80
60
“chase”
40
“normal” labeling
20
0
0
10
© 2002 Waterborg - UMKC
20
30 min
60
Steady-State Labeling Protocol
% specific label
exponential rise of specific label
to steady-state condition maximum
100
steady-state
50
Mathematical fit of data
to “exponential rise
to maximum” yields
T1/2 half-life value.
half-life
T1/2
"continuous label"
0
0
10
20
30
40
50
time
© 2002 Waterborg - UMKC
60
70
80
90 100
*Acetate Labeling in Yeast
cpm/prep
HPLC data
cpm/peak
non-histone protein
HPLC peak
(at 95 min)
15000
15,000,000
total
*Ac label
10000
10,000,000
5,000,000
5000
histone“steady-state”
labeling:
apparent "steady-state"
0
0
10
20
30
40
min
© 2002 Waterborg - UMKC
50
60
0
70
Half-life Measurement in Yeast H4
Specific radioactivity
Coom
Histone H4
HPLC
1500
gel
1.5
min with *Ac (+chx)
-chx
2 5 10 15 20 3150 71 100
1000
1.0
500
0.5
0.0
0
0
98-day fluorograph
© 2002 Waterborg - UMKC
30
60 min
Half-life Measurement in Yeast H3
Specific radioactivity
Coom
Histone H3
min with *Ac (+chx)
-chx
2 5 10 15 20 31 50 71 100
gel
1.5
HPLC
1000
1.0
500
0.5
0.0
0
0
98-day fluorograph
© 2002 Waterborg - UMKC
30
60 min
Acetylation Turnover in Yeast Histones
Turnover rates of post-translational acetylation
in Saccharomyces cerevisiae
histone
half-life in
YPD medium
half-life in
SD medium
H4
H3
21 ± 5 8 ± 2
H2B H2A
4±1 6±2
14 ± 1 12 ± 4 4 ± 1 5 ± 3
© 2002 Waterborg - UMKC
Turnover across Acetylated H4 Forms
H4 acetylation turnover in YPD
35
H4
half-life (min)
30
25
Conclusion:
Multi-acetylated forms of
yeast histone H4 turn
over faster than
mono-acetylated forms.
20
average
half-life
15
10
5
0
1
2
3
4
# AcLys per H4
© 2002 Waterborg - UMKC
Turnover across Acetylated H3 Forms
H3 acetylation turnover in YPD
20
half-life (min)
H3
15
Conclusion:
Rates of turnover are the
same for mono- up to
penta-acetylated forms in
yeast histone H3.
regression
fit
10
5
0
1
2
3
4
5
# AcLys per H3
© 2002 Waterborg - UMKC
Acetylation Turnover in Chlamydomonas
Add chx at t = –15 min
Add 200 M NH4Ac at t = –5 min
Add 10 M NaAc (2 mCi [3H]-Ac)
at t = 0 min
Add chx at t = –10 min
Add 10 M NaAc (2 mCi
[3H]-Ac) at t = 0 min
label (%)
hplc
label (%)
100
100
gel
80
60
50
40
20
0
0
0
10
20
30 min 60
0
10
data for H2B
© 2002 Waterborg - UMKC
20 min 30
Comparing Histone Acetylation Turnover
Histone:
H4
H3
H2B
H2A
Chlamydomonas
(algae)
3.5 ± 1.1
1.7 ± 0.2
1.4 ± 0.3
2±1
Saccharomyces
(yeast-fungi)
~17
~10
4
6
30 ± 11
35 ± 13
26 ± 9
28 ± 5
—
Half-life (min)
Turnover half-life (min)
Medicago
H3.1
(alfalfa-plant) H3.2
4
Chlamydomonas H3
3
2
1
0
1
2
© 2002 Waterborg - UMKC
3
4
5 #Ac/H3
Steady-state Specific Labeling of H3
4
4
5
3
label
3
2
2
1
1
time
Standard label at "max"
0
5
4
3
2
1
0
© 2002
Waterborg
#Ac/H3
1
2- UMKC 3
4
5
#Ac/H3
Turnover
5
82 ± 17 %
4
106 ± 10 %
3
104 ± 20 %
2
92 ± 9 %
1
100 ± 17 %
H3 total
100 ± 12 %
Extent of Acetylation Turnover
5
Chlamydomonas H4
Standard label at "max"
Standard label at "max"
Chlamydomonas H3
100%
4
3
2
1
0
3
4
3
<50%
2
1
Yeast H3
100%
4
~50%
2
1
0
#Ac/H3 1
#Ac/H4 1
5
Standard label at "max"
Standard label at "max"
2
5
3
100%
4
0
#Ac/H3 1
6
5
5
2
4
Yeast H3
100%
4
3
3
<40%
2
1
0
2
3
© 2002 Waterborg - UMKC
4
5
#Ac/H4
1
2
3
4
5
Conclusions
1. Measure dynamic acetylation by steady-state labeling in vivo.
2. Histone acetylation half-lives per acetylation level.
3. Information about chromatin environments from whole-cell studies.
© 2002 Waterborg - UMKC
Thank you for your attention
© 2002 Waterborg - UMKC