Chromatin remodelling ATPase Brg-1 is an essential factor for the maintenance of the intestinal crypt stem cell and adenoma

formation

Aliaksei Holik

BRG-1 and cancer

BRG-1 mutations are found in human cancer cell lines and in primary cancers

Heterozygous deletion of Brg-1 in mice promotes tumourigenesis via Brg-1 haploinsufficiency

Restoration of BRG-1 function leads to the reversion of a cancer phenotype

c-Fos

BRG-1

BRCA1 RB/p21

p53

Wnt-pathway

Suggested mechanisms of BRG1 role in 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

Cre recombinase Ah ER Cre recombinase Villin ER

BRG1 loxP loxP

BRG1 loxP loxP

Brg-1-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

Ratio

of r

ecom

bine

d cr

ypts

Day 3 Day 5 Day 7BRG-1

AhCre-ERT

Brg-1-deficient small intestinal epithelium appears to be normal at day 4 post induction

Brg1fl/fl

Control

BRG-1

VillinCre-ERT

Brg1-deficient small intestinal epithelium displays no difference in mitosis and

apoptosis levels

Frequency of Mitosis and Apoptosis Per Crypt

0

0.2

0.4

0.6

0.8

1

1.2

Mitosis Apoptosis

VillinCre-Brg1fl/fl

VillinCre+Brg1fl/fl

p-values:

Mitosis – 0.629

Apoptosis – 0.057

Brg1-deficient small intestinal epithelium displays no difference in mitosis and

apoptosis levels

VillinCre-Brg1fl/fl

VillinCre+Brg1fl/fl

Apoptosis Position Distribution

0

0.5

1

1.5

2

2.5

3

3.5

1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 25-27 28-30 31-33

Cell Position

Inactivation of Brg-1 in small intestinal epithelium leads to rapid crypt loss

Brg1fl/fl

Control

Day 5 Day 7Day 4

Brg-1 loss leads to the failure of the crypt stem cell

Active stem cell

Brg1+ Brg1-Brg1-Transit amplifying cells

Relative Quantity of Ascl2 and Lgr5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Ascl2 Lgr5

Loss of Brg-1 results in reduced expression of intestinal stem cell markers

VillinCre-Brg1fl/fl

VillinCre+Brg1fl/fl

p-value < 0.05

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

Brg-1/Apc double mutant cells are selectively eliminated in small intestine

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

BRG-1-catenin

Brg-1-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

Brg-1 deletion removes a portion of Apc-deficient cells decreasing tumour

burden

Brg1-/Apc-

Brg1- Apc-

Brg-1 deletion removes a portion of Apc-deficient cells decreasing tumour

burden

Brg1-/Apc-

Brg1- Apc-

Microadenoma

Adenoma

Stem cell

Transit amplifying cells

Adenoma progression depends on the cell of origin

Conclusions

Brg-1 is a crucial factor for the maintenance of the adult tissue stem cell in small intestine.

Brg-1 loss is incompatible with activation of the Wnt pathway in the murine small intestine

Targeting the intestinal stem cell provides 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