Molecular Endocrinology of Obesity and Diabetes

Regulation of Gene Expression in the Endocrine Pancreas

Stein LectureMonday January 18, 2010

Analysis of Distal ‘Enhancer’ Control Regions

1) ‘Localization’a) Sequence conservationb) Epigeneticsc) DNA-protein footprinting

2) Functiona) Reporter-driven transfection assays (in vitro)b) Transgenic Assays (in vivo)c) Targeted deletion of control region (in vivo)

3) Dissectiona) ‘Element’ based mutational analysisb) Element based DNA-protein gel shiftsc) Identity of protein(s) binding factord) ChIP

Factor

PDX-1

BETA2

MafA

PAX-6

Distribution

β and δ cells, exocrine pancreas,stomach, duodenum

all islet cell types, intestine, brain

only islet β cells,lens, neural tube

all islet cell types,eye, CNS

Null Phenotype

Apancreatic

Reduced β cell numbers,no mature islets, diabetic

Adult phenotype, reduced β cell numbers, diabetic

reduced β cell numbers,No α cells, diabetic

Targets

Ins, IAPP, GK, PDX-1

Ins, IAPP, GK

Ins, PDX-1

Ins, IAPP, GK, PDX-1

+1

A1A3 C1C2 E1

PAX-6 PDX-1BETA2/

E47 PDX-1

-350

MafA

INS

Transcriptional regulation of Insulin is mediated by factors important in β cell development and function

Models depicting the arrangement of factors on chromatin in acute glucose without and with IL-1 and under hyperglycemic conditions in the presence of IL-1.

Lawrence M C et al. PNAS 2009;106:22181-22186

©2009 by National Academy of Sciences

PDX-1 Distribution in Adult Islet

cell

cell

cell

PP cell

CORE MANTLE

<100%

9%

15%

3%

i

Wu KL, et al.; Mol Cell Biol. 1997

Transcription factors and pancreas development

Gene inactivation effects:

Delete pancreatic buds

Delete/reduce specificislet cell types

Pdx1, PTF1a/p48, Isl1(d), Hlxb9

Pax4, Pax6, Arx1, Nkx2.2, Nkx6.1,NeuroD/BETA2, MafB

Isl1(v), Ngn3, HNF6

Delete islet endocrine cells

MODY4 (Pdx1)

MODY6 (BETA2)

MODY1 (HNF4)

MODY2 (glucokinase)

MODY3 (HNF1)

MODY5 (HNF1)

Known mutations leading to MODY in humans

MODY= maturity onset diabetes of the young (monogenic dominant form of Type 2 diabetes)

HNF6

HNF3

Pdx1

HNF4

HNF1

ngn3 Beta2/NeuroD1

insulin(MODY4)(MODY3)

(MODY1) glucokinase(MODY2)

(MODY5)HNF1

(MODY6)

Interactions of known MODY genes

transcription factorsglucose metabolism

GLUT2

Functional assignments for Pdx-1 target genes

Gene description I. Cell adhesion Cdh24 Cadherin-like 24 Negr1 Neuronal growth regulator 1 Pak1 p21-activated kinase 1 Parvb Parvin, II. Cell cycle Anapc5 Anaphase-promoting complex 5 Ccnb1 Cyclin B1 Ccrk Cell cycle-related kinase Cdk8 Cyclin-dependent kinase 8 III. Cell growth/death Bcl7b B-cell CLL/lymphoma 7B Casp3 Caspase 3 Fadd Fas-associated via death domain St18 Suppression of tumorigenicity 18 IV. Cytoskeleton Epb4.1 Erythrocyte protein band 4.1 Mark2 MAP affinity-regulating kinase 2 Myh9 Myosin, heavy polypeptide 9 Pfn2 Profilin 2 Sntb2 Syntrophin, basic 2 Sntg1 Syntrophin, 1 V. Exocytosis Pclo Piccolo Rph3a Rabphilin 3A Syn1 Synapsin I Syt7 Synaptotagmin VII Syt11 Synaptotagmin XI Syt13 Synaptotagmin XIII VI. Hormone processing Pcsk1 Proprotein convertase 1 Pcsk2 Proprotein convertase 2

VII. Intracellular trafficking

Arfl4 ADP-ribosylation factor 4-like Chm Choroidermia Ica1 Islet cell autoantigen 1 Rab10 RAB10, member RAS family Rab21 RAB21, member RAS family Rab3ip RAB3A interacting protein Tom1l2 Target of myb1-like 2 Trappc2 Trafficking protein particle complex 2 Vamp8 Vesicle-associated membrane 8 Vapb VAMP-associated protein B and C VIII. Metabolism Atp5b ATP synthase, F1 complex, Atp5g2 ATP synthase, F0 complex, c2 Eno1 Enolase 1 G6pc2 Glucose-6-phosphatase, catalytic, 2 Glud1 Glutamate dehydrogenase 1 Gyk Glycerol kinase Hmgcr HMG-coenzyme A reductase Mdh1 Malate dehydrogenase 1 Ndufb8 NADH dehydrogenase 1, 8 Pccb Propionyl-CoA carboxylase, Pgam1 Phosphoglycerate mutase 1 Pla2g6 Phospholipase A2, group VI Txn1 Thioredoxin 1 IX. Nuclear Crsp2 Cofactor required for Sp1, subunit 2 Hes6 Hairy and enhancer of split 6 Id3 Inhibitor of DNA binding 3 Isl1 Islet-1 Klf7 Kruppel-like factor 7 Mybl2 Myeloblastosis oncogene-like 2 Myst2 MYST histone acetyltransferase 2 Myt1 Myelin transcription factor 1 Neurod1 Neurogenic differentiation 1 Nkx2-2 NK2 transcription factor related Pax6 Paired box gene 6 Pbx1 Pre-B-cell leukemia factor 1 Pdx-1/Ipf1 Insulin promoter factor Trp53 Transformation related protein 53

X. Signal transduction Frap1 FK506-binding protein 12-rapamycin associated protein 1 Fyn Fyn proto-oncogene Il1r1 Interleukin 1 receptor, type I Impa1 Inositol (myo)-1(or 4)-monophosphatase 1 Inpp5f Inositol polyphosphate-5-phosphatase F Pde4b Phosphodiesterase 4B Pde10a Phosphodiesterase 10A Pi4k2b Phosphatidylinositol 4-kinase 2b Pik3c2g Phosphatidylinositol 3-kinase, C2 domain, Pik4cb Phosphatidylinositol 4-kinase, catalytic, Plcl3 Phospholipase C, 1 Ppp2r2c Protein phosphatase 2, regulatory subunit B, Prkca Protein kinase C Prkce Protein kinase C Psen2 Presenilin 2 XI. Transporter activity Abcc8 ATP-binding cassette, subfamily C, member 8 Atp6v0a1 ATPase, H+ transporting, lysosomal V0 subunit A1 Cacna1c/Cav1.2 Calcium channel, voltage-dependent, L type, 1C subunit Cacna1h/Cav3.2 Calcium channel, voltage-dependent, T type, 1H Kcnj11/Kir6.2 Potassium inwardly rectifying channel, subfamily J, member 11 Slc2a3 Facilitated glucose transport

e8.5 e13.5

adulte9.5

PDX-1

gut endoderm

ventral pancreasdorsal pancreas

endocrine islets: and PP

Ahlgren U, et al.; Genes Dev. 1998

Throughout pancreatic duct & in small clusters

Pancreatic-Duodenal Homeobox-1 expression earliest identified islet specific factor involved in pancreas development

PDX-1 is required for proper outgrowth of pancreas and differentiation of rostral

duodenum

Absence of pancreatic tissuedie postnatally / hyperglycemic

Inhibition of gastric emptyingStomach/duodenal junction malformation

Offield MF, et al.; Development. 1996

Pancreas Development

Islet Cell Progenitors

Maintenance of cell function in adult islets of mice and humans

(MODY4)

Understanding the transcriptional regulation mediating Pdx-1 tissue specific expression will likely

provide information relevant to push stem cells towards the ‘islet’ fate

for use in transplantation.

Proper Expression of PDX-1 Plays an Important Role In:

-2560 -1880

1-1330 -800

2-260 +180

3

ATG

pdx-1

-2761 -2457

I

-2153 -1923

II

-1879 -1608

III

HSS 1

Conserved Sequences Upstream of the Promoter Region are Important in

Regulation of pdx-1

pdx-1

I II III

Area IV

FoxA2Nkx2.2/PDX-1

Area IV is a Conserved Regulatory Domain of pdx-1

-6.5 -3.0

catTK

non-

Gerrish K, et al. Mol Endo. 18(3):2003

Transfections:

Transgenics:

1 160 480 772 992-2917/-1918 100%

Area II

Area I (m160-480) 20

Inactive

Area I /Area II

30

100~500bp

Area I Sequences Potentiate an Area II Transgene Throughout the Cell

Population

e13.5

PDX-1

Glucagon

Insulin

e14.5 e15.5

AI/IIAI/II AI/II

AI/AII are Sufficient to Regulate Expression in Mature -Cells

-gal InsulinNuclei

AI/II+/insulin -

-gal SomatostatinNuclei

AI/II+/somatostatin +

-cells

-cells

-cells

Insulin+ 100% 35% 98%

Somatostatin+ 15% NA 12%

Glucagon+ 3% — —

PP+ 9% NA —PP-cells

PDX-1+ AII+ AI/II+

AI/AIILacZ is Expressed in Islet Cells, and Cells

HNF1

Pax-6

pdx-1

PDX-1 MafAFoxA2 FoxA2

Area I Area II

Nkx2.2

Area I Stimulation of Area II is Mediated by -cell Enriched

Factors

I II

duodenal/pancreatic precursors

pancreatic precursorsduodenal mucosa &enteroendocrine cells

endocrine cells exocrine cells

PP

endoderm

induce Pdx1 (Areas I-IV)

Pdx1 (Areas I-III)induced by Ptf1a in Area III

decreased Pdx1 Pdx1Hi (Areas I/II)

Pdx1+

loxP loxP

E1 E2pdx-1

I-II-III

loxP

islet cell‘enhancer’ cell

‘enhancer’

cross-species sequence conservation

Y. Fujitani, M. Gannon & C. Wright

pdx-1

~90%~30%~30%

(~1kb @ -2 kb)

rpl21 60Sgsh1(-200 kb/-100 kb)

cdx2+40 kb

Much strong evidence that “lots of the action” converges on the conserved Area I-II-III

loxP loxP

E1 E2pdx-1

I-II-III

loxP

islet cellenhancer acinar cell

enhancer

cross-species sequence conservation

Y. Fujitani, M. Gannon; R. Stein

pdx-1

~90%~30%~30%

(~1kb @ -2 kb)

rpl21 60Sgsh1(-200 kb/-100 kb)

cdx2+40 kb

Much strong evidence that “lots of the action” converges on the conserved Area I-II-III

conservedarea I-II-III

WTE1 E2

[I-II-III]

loxP

global deletion

flox[I-II-III]

loxPloxP

ongoing tissue-specific deletion experiments

normal function

E1 E2

E1 E2

Yoshio Fujitani

hypomorphic allele

Pdx1I-II-III allele “”

Lower protein levels than WT.Incorrect spatiotemporal expression pattern in pancreas primordia.

Deleting Pdx1 function causes absence of pancreas, and gut defects…

What happens when function is reduced?

Different threshold requirements of foregut & pancreas progenitors to Pdx1

• transcriptional networks (complexes, titers), link to cell differentiation • intercellular signaling inputs on Pdx1

………goals for these long-term in vivo studies…

Effect of Pdx1 downregulation vs. absence in specific progenitors?

But, how to define progenitor cell types (genetics, markers)?[hurdles: non-stereotypic outgrowth, marker paucity]

Dissect Pdx1 enhancer(s) into defined functional motifs

loxP loxP

E1 E2pdx-1

I-II-III

loxP

mammal-specific?

• BAC recombineering• RMCE/Cassette Acceptor

MODY factors

[HNF1, PDX1]

Foxa2, KLF11, Pax6,

MafA, HNF6

HNF6 required for:• pancreas outgrowth (phenotype similar to Pdx1/)

• Ngn3-based endocrine commitment