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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