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