Chromatin remodelling ATPase Brg1 is an essential factor for the maintenance of the intestinal crypt stem cell and adenoma

formation

Aliaksei Holik

DNA is packed into Chromatin

Nicolas Bouvier, Curie Institute, Paris, France

Chromatin remodelling complexes enable access for

DNA binding proteins

Transcription factors

OFF

ON

Chromatin remodelling

complex

ATP

BRG1 and cancer

BRG1 mutations are found in cancer cell lines and in primary cancers

Heterozygous deletion of Brg1 in mice promotes tumourigenesis due to Brg1 haploinsufficiency

Restoration of BRG1 function leads to the reversion of a cancer phenotype

c-Fos

BRG1

BRCA1 RB/p21

p53

Wnt-pathway

Suggested mechanisms of BRG1 mediated tumourigenesis

Wnt pathway

Willert K., Jones K. A. Genes Dev. 2006;20:1394-1404

Small intestine architecture

Villus

Crypt

Stem cell

Transit amplifying cells

loxP strategyGeneration of conditional knock-out using Cre-loxP techniques

X BRG1

Cre recombinase Ah

loxP loxP

Mouse 1 Mouse 2

Cre recombinase Ah

BRG1

loxP loxP

Cre recombinase Ah

ER

ER

ER

Brg1-deficient cells in small intestine are repopulated with wild-type cells

Timeline of gut repopulation

0

0.1

0.2

0.3

0.4

0.5

0.6

3 5 7 10 14

Days Post Induction

Rat

io o

f re

com

bin

ed c

ryp

ts

Day 3 Day 5 Day 7

Brg1-deficient small intestinal epithelium appears to be normal at day 4 post induction

Brg1fl/fl

Control

-BRG1

Brg1-deficient small intestinal epithelium displays lower apoptosis levels

Apoptosis per Crypt

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Control Brg Hom

BrdU Positive Cells Per Half Crypt

0.00

2.00

4.00

6.00

8.00

10.00

12.00

Control Brg Hom

Inactivation of Brg1 in small intestinal epithelium leads to rapid crypt loss

Brg1fl/fl

Control

Day 3 Day 5 Day 7

Brg1 deletion leads to the failure of the crypt stem cell

Active stem cell

Brg1+ Brg1+Brg1-

Generation of double knock-out using Cre-loxP techniques

X BRG1

Cre recombinase Ah

loxP loxP

Mouse 1 Mouse 2

Cre recombinase Ah

BRG1

loxP loxP

Cre recombinase Ah

ER

ER

ER

APC

loxP loxP

APC

loxP loxP

Brg1/Apc double mutant cells are selectively eliminated in small intestine

Small intestine of Brg1fl/fl/Apcfl/fl mouse at day 7 post induction

-BRG1--catenin

Brg1-deficiency rescues lethal phenotype of Apc deletion in double knock-out mice

Control

Brg1fl/flAPCfl/fl

APCfl/fl

Brg1fl/fl

Median survival APCfl/fl=8 Brg1fl/flAPCfl/fl=14, P=0.0010

Brg1 removes a portion of Apc-deficient cells decreasing tumour burden

Brg1-/Apc-

Brg1- Apc-

Brg1 removes a portion of Apc-deficient cells decreasing tumour burden

Brg1-/Apc-

Brg1- Apc-

Microadenoma

Adenoma

Stem cel

l

Transit amplifying cells

Adenoma progression depends on the cell of origin

Conclusions

Brg1 is a crucial factor for the maintenance of the adult tissue stem cell in small intestine. This is the first evidence of Brg1 requirement for the somatic stem cell maintenance

Targeting the active sub-population of intestinal stem cells provide an attractive concept for the removal of potentially malignant lesions in individuals with genetic predisposition to colorectal cancer

Acknowledgements

The Darwin Trust of Edinburgh

Alan Clarke

Boris Shorning

ARC group