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The importance of topoisomerasesfor chromatin regulated genesAARHUS UNIVERSITY
Fredsoe J, Pedersen JM, Roedgaard M, Andersen AHLaboratory of Genome Research, Department of Molecular Biology and Genetics, University of Aarhus, Denmark.
DNA topoisomerases are enzymes, which function to relieve torsional stress in the DNA helix by introducing transient breaks into the DNA molecule. By use of Saccharomyces cerevisiae and microarray technology we have previously shown that topoisomerases are required for the activation of chromatin regulated genes. A further study of the PHO5 gene belonging to this gene class demonstrated a lack of binding of the transcription factor Pho4 to the PHO5 promoter in the absence of topoisomerases, most likely due to changes in the overall superhelical state of the promoter.The GAL genes also require topoisomerases for optimal activation, but in contrast to the PHO5 gene, topoisomerases are not required for chromatin remodeling of the GAL1/10 promoter region, indicating a different role of the enzymes. We are currently performing a detailed investigation of the GAL genes to elucidate the precise role of topoisomerases during the activation process, and the obtained results will be presented.
ABSTRACT
INTRODUCTION
Transcription elongation generates negative and positive supercoiling in front of and behind the RNA polymerase, respectively.
In the absence of functional topoisomerases in S. cerevisiae, a large percentage of genes are de-regulated 2-fold or more. This de-regulation correlates with transcriptional activity (A), but not transcript length (B). Furthermore, genes de-regulated by lack of topoisomerases show high sensitivity to chromatin regulation (C).
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
-4 -2 0 2 4Transcriptional activity (log2)
top1Δtop2ts
Gen
e ex
pres
sion
ch
ange
s (lo
g2)
Transcript length (nt)
top1Δtop2ts
Gen
e ex
pres
sion
ch
ange
s (lo
g2)
-1
-0.8-0.6
-0.4-0.2
00.2
100 1000 10000-1
-0.8-0.6
-0.4-0.2
00.2
100 1000 10000-0.2
-0.1
0.0
0.1
0.2
-2 -1 0 1
Sens
itivi
ty to
chr
omat
in
regu
latio
n (lo
g2)
top1Δtop2ts gene expression changes (log2)
DNA katenering
DNA polymerase DNA polymerase RNA polymerase
RNAPII
RNA polymerase
DNA topoisomerases
OverwindingUnderwinding
“Top1”-DNA kløvningskompleks
DNA topoisomerase II/RecQ helicase
Replikation (A) Transkriptions-koblet repair (C)
DNA polymerase
DNA topoisomeraser
A B C
THE GAL GENES
In the absence of glucose and galactose, the promoters of the GAL genes are found in a poised state, where the transcription factor Gal4 is bound to the promoter along with Gal80. When galactose becomes available, Gal80 will dissociate and allow Gal4 to recruit chromatin remodelers, which in turn exposes the TATA box, so that the TATA box binding protein (TBP) can bind and facilitate the formation of the general transcription machinery (A, B).
Topoisomerases are required for optimal transcriptional activation of GAL genes (C) but not for nucleosome eviction (D).Topoisomerases are dispensable in the absence of Gal80, suggesting that the enzymes are required only during gene activation (E).
Gal2p
galgal
gal
gal
gal
galgal
Gal80pGal80p
Gal3pGal3p
gal
Gal3pGal3p
Gal80pGal80p
Gal80pGal80p
Gal80pGal80p
Gal4p Gal4pGal4p Gal4p
Gal80pGal80p
Gal4p Gal4pgluglu
Galactose
Poised state(Glycerol, raffinose)
Repressed state(Glucose)
Active state(Galactose)
ChromatinremodelersGeneral
transcriptionfactors
Gal1pgalactose-1p
glucose-1p
glucose-6pglycolysis
Gal5p
Gal7pGal10pUDP-glu
UDP-gal
Gal1p
Gal1p only when induced.Binding of Gal1p resultsin higher affinity for galactose
Mig1p Mig1pIn glucose Mig1p, Mig2p, and Mig3pare importet into the nucleus and bindat the GAL promoter (1, 2, and 3)
Gal2p is a galatose permadasewhich transports galatose intothe cell
Gal3p share ~70% homology with Gal1p and are able to bind galactose. GAL3 null strains takes 3-4 days to induce the GAL genesThe chemistry of galatose’s
entry into the glycolysis and the enzymes involved (10) When Gal3p has bound galatose
it is able to interact with Gal80p,in an ATP dependent way. Thiswill keep Gal80p in the cytoplasm
In the absence of galactoseGal80p binds as a dimer to Gal4p, thus preventing Gal4p in recruitingother factors Active Gal4p recruits a
variety of activating factors
0,01
0,1
1
10
100
1000
0 20 40 60 80 100 120
mRN
A le
vels
(%)
Time after induction (minutes)
GAL1
wild type gal80∆ gal80∆top1∆top2ts
0,01
0,1
1
10
100
1000
0 20 40 60 80 100 120Time after induction (minutes)
GAL10
Galactose(activation)
GAL7 ORF GAL10 ORF GAL1 ORF
Gal4pconcensus
Gal4pconcensus
TATA box TATA box TATA box
GAL7 ORF GAL10 ORF GAL1 ORF
Gal4pconcensus
Gal4pconcensus
TATA box TATA box TATA box
0,0
20,0
40,0
60,0
80,0
100,0
120,0
0 30 90
GAL1
0,0
20,0
40,0
60,0
80,0
100,0
120,0
0 30 90
GAL2
0,0
20,0
40,0
60,0
80,0
100,0
120,0
0 30 90
GAL7
0,0
20,0
40,0
60,0
80,0
100,0
120,0
0 30 90
GAL10
0
0,2
0,4
0,6
0,8
1
1,2
0 30 90
GAL2
0
0,2
0,4
0,6
0,8
1
1,2
0 30 90
GAL7
0
0,2
0,4
0,6
0,8
1
1,2
0 30 90
GAL1
0
0,2
0,4
0,6
0,8
1
1,2
0 30 90
GAL10
-3,5-3
-2,5-2
-1,5-1
-0,50
0,51
1,5
278600 278800 279000
GAL1
-2,5-2
-1,5-1
-0,50
0,51
1,5
289850 290050 290250 290450
GAL2
-3,5-3
-2,5-2
-1,5-1
-0,50
0,51
1,5
275300 275500 275700 275900
GAL7
-3,5-3
-2,5-2
-1,5-1
-0,50
0,51
1,5
278250 278450 278650
GAL10
Perc
enta
ge in
duct
ion
Induction time (minutes)Induction time (minutes)
H3
enric
hmen
t
mRNA levels H3 ChIP Primer placement
Nuc
leos
ome
occu
panc
y
Chromosomal coordinates
wild type top1∆top2ts
A
C DE
B
MODELAfter successful binding of transcription factor (1), chromatin remodelers are recruited (2) facilitating nucleosome eviction (3). Exposure of the TATA box allows for binding of the TATA box binding protein (TBP)(4), which will recruit the general transcription machinery (5), allowing active transcription (6).Topoisomerases can be required for initial transcription factor binding (in the case of PHO5, step 1), or in the binding of TBP (GAL genes, step 5).Future investigation of promoter superhelicity combined with studies of TBP binding will be used to assess the validity of the model.
Transcription factorbinding
Recruitment of chromatinremodelers
Ac Ac
AcAc
AcAc
Histone evictionTPB recruitment
Transcription complexrecruitment/formation Active transcription
1 2
3 4
5 6
THE PHO5 GENE
In the absence of functional topoisomerases, the phosphate regualted gene PHO5 is unable to undergo transcriptional activation (A). However, if PHO5 is constitutive expressed, topoisomerases become dispensable for continued transcription (B).
The PHO5 promoter is covered by four positioned nucleosomes (C). These nucleosomes are removed following binding of the transcription factor Pho4. Without topoisomerase activity the nucleosomes remain bound to the promoter (D), due to lack of Pho4 binding (E).
Time after phosphate depletion (min)
0
2
4
6
8
10
12
0 45 90 135 180
wild typepho80Δpho80Δtop1Δtop2ts
mR
NA
leve
ls (l
og2)
252
Time after phosphate depletion (min)
wild typetop1Δtop2tstop1Δtop2ts
450
20
40
60
80
100
120
0 90 135 180
mR
NA
leve
ls (%
) 232
00.20.40.60.8
11.21.4
0 45 90 135 180
His
tone
H3
enric
hmen
t
wild typetop1Δtop2ts
Time after phosphate depletion (min)
-1
-0.5
0
-4 -3 -2 -1
UAS1
-750 -600 -450 -300 -150 0Distance from PHO5 bp)
Nuc
leos
ome
occu
panc
y (lo
g2)
TSS (
-4 -3 -2 -1
UAS2 TATA
PHO5
0
1
2
3
4
5
6
0 45 90 135 180
Pho
4-13
xcM
yc e
nric
hmen
t wild type top1Δtop2ts
Time after phosphate depletion (min)
A B
C D E