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![Page 1: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/1.jpg)
Molecular Endocrinology of Obesity and Diabetes
Regulation of Gene Expression in the Endocrine Pancreas
Stein LectureMonday January 18, 2010
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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
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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
![Page 4: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/4.jpg)
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
![Page 5: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/5.jpg)
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
![Page 6: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/6.jpg)
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
![Page 7: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/7.jpg)
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)
![Page 8: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/8.jpg)
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
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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
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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
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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
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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:
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-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
![Page 14: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/14.jpg)
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
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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
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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
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-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
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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
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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+
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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
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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
![Page 22: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/22.jpg)
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
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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?
![Page 24: Molecular Endocrinology of Obesity and Diabetes Regulation of Gene Expression in the Endocrine Pancreas Stein Lecture Monday January 18, 2010.](https://reader033.fdocuments.in/reader033/viewer/2022061522/56649ec95503460f94bd7229/html5/thumbnails/24.jpg)
Different threshold requirements of foregut & pancreas progenitors to Pdx1
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• 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
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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