The Role of Hepatic Nuclear Factor 1α and PDX-1 in Transcriptional ...

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1 JBC M1:03538 REVISED 9/14/01 The Role of Hepatic Nuclear Factor 1α and PDX-1 in Transcriptional Regulation of the pdx-1 Gene Kevin Gerrish 1 , Michelle A. Cissell 1 , and Roland Stein 1,2,3 1 Department of Molecular Physiology and Biophysics Vanderbilt University Medical Center Nashville, TN 37215 2 Corresponding author Phone: (615) 322-7026 Facsimile: (615) 322-7236 Email: [email protected] Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on October 5, 2001 as Manuscript M109244200 by guest on March 27, 2018 http://www.jbc.org/ Downloaded from

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JBC M1:03538 REVISED 9/14/01

The Role of Hepatic Nuclear Factor 1α and PDX-1 in

Transcriptional Regulation of the pdx-1 Gene

Kevin Gerrish1, Michelle A. Cissell1, and Roland Stein1,2,3

1 Department of Molecular Physiology and Biophysics

Vanderbilt University Medical Center

Nashville, TN 37215

2 Corresponding author Phone: (615) 322-7026

Facsimile: (615) 322-7236

Email: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 5, 2001 as Manuscript M109244200 by guest on M

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ABSTRACT

The PDX-1 homeodomain transcription factor regulates pancreatic development

and adult islet β cell function. Expression of the pdx-1 gene is almost exclusively

localized to β cells within the adult endocrine pancreas. Islet β cell-selective transcription

is controlled by evolutionarily conserved subdomain sequences (termed Area I (-2839 to -

2520 base pairs (bp)), II (-2252 to -2023 bp), and III (-1939 to –1664 bp)) found within

the 5’-flanking region of the pdx-1 gene. Area I and Area II are independently capable of

directing β cell-selective reporter gene activity in transfection assays, with Area I

mediated stimulation dependent upon binding of hepatic nuclear factor 3β (HNF3β), a

key regulator of islet β cell function. To identify other transactivators of Area I, highly

conserved sequence segments within this subdomain were mutagenized and their effect

on activation determined. Several of the sensitive sites were found by transcription

factor database analysis to potentially bind endodermally expressed transcription factors,

including HNF1α (-2758 to -2746 bp, Segment 2), HNF4 (-2742 to -2730 bp, Segment 4;

-2683 to -2671 bp, Segment 7-8), and HNF6 (-2727 to -2715 bp, Segment 5). HNF1α,

but not HNF4 and HNF6, binds specifically to Area I sequences in vitro. HNF1α was

also shown to specifically activate Area I driven transcription through Segment 2. In

addition, PDX-1 itself was found to stimulate Area I activation. The chromatin

immunoprecipitation assay performed with PDX-1 antisera also demonstrated that this

factor bound to Area I within the endogenous pdx-1 gene in β cells. Our results indicate

that regulatory factors binding to Area I conserved sequences contribute to the selective

transcription pattern of the pdx-1 gene, and that control is mediated by endodermal

regulators like HNF1α, HNF3β, and PDX-1.

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INTRODUCTION

Several transcription factors that are enriched within pancreatic islet cells mediate

differentiation during embryogenesis and the maintenance of specialized cellular

functions in the adult (1-3). Some of these proteins are also dysfunctional in patients

with diabetes, a disease caused in part by the failure of β cells to produce sufficient

insulin to meet the body’s needs (4-6).

The PDX-1 (Pancreas Duodenum homeobox-1; also known as STF-1, IDX-1,

IPF-1, IUF-1) transcription factor is an essential regulator of pancreatic development and

adult islet β cell function (7-10). In the pancreas, PDX-1 is expressed almost exclusively

in islet β cells (11) and regulates transcription of the genes associated with β cell identity,

including insulin (12-16), glucokinase (17), islet amyloid polypeptide (18-21), and

glucose transporter type 2 (GLUT2) (22). Selectively removing PDX-1 from islet β cells

in mice compromised their ability to maintain glucose homeostasis and resulted in the

development of diabetes, at least partially due to reduced β cell expression of insulin and

GLUT2 (23). In addition, mice that are heterozygous mutant carriers of PDX-1 exhibit

signs of glucose intolerance, suggesting that pdx-1 gene dosage affects insulin expression

and β cell function (23,24). Humans with this condition are susceptible to different forms

of non-insulin dependent diabetes, including mature onset diabetes of the young (MODY)

(25-27).

Pancreatic development and islet β cell function also are influenced by hepatic

nuclear factor (HNF) 1α (28-30), 1β (31), 3β , 4α (32-34), and 6 (35). For instance,

HNF1α-/- (30) and HNF6-/- (35) mice exhibit a non-insulin dependent diabetic phenotype

which is associated with β cell dysfunction. Heterogeneous nonfunctional mutations in

HNF1α (29), HNF1β (31), and HNF4α (34) are also found in human MODY patients.

Each distinct HNF transcription factor appears to be required for controlling key

differentiation and metabolic programs in the pancreas and liver (30, 35-38).

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The sequences that control appropriate developmental and adult specific

expression of the pdx-1 gene were demonstrated in transgenic studies to be located in the

5’-flanking region of the mouse (39, 41) and rat (40) genes. Three nuclease

hypersensitive sites, termed HSS 1 (-2560 to -1880 bp), 2 (-1330 to -880 bp), 3 (-260 to

+180 bp), were identified within this region of the endogenous mouse gene by DNase I

and micrococcal nuclease analysis (39). However, only HSS1 sequences were capable of

directing pancreatic β cell-selective expression in transgenic and transfection studies (39).

Sequence analysis of the mouse, chicken, and human pdx-1 genes revealed that the HSS1

region also represented the only area of significant identity within 4.5 kilobases of the

transcription start site (41). Sequence conservation and function allowed the HSS1 region

to be divided into the Area I (-2839 to -2520 bp), Area II (-2252 to -2023 bp) and Area III

(-1939 to –1664 bp) subdomains (41). Areas I and II, but not III, were capable of

independently directing β cell-selective reporter gene activity in transfection assays, with

Area I activation mediated by HNF3β (41). This forkhead transcription factor is also

necessary for stimulation by Area II, although only in the context of both Area I and II

sequences (39,41). The importance of HNF3β in pdx-1 transcription was also supported

by results showing that pdx-1 mRNA levels were reduced in homozygous null HNF3β

embryoid bodies (41).

Collectively, the data obtained from analysis of transgenic and transfected pdx-1

driven reporter constructs strongly suggested that the HSS1 region played an essential

role in directing transcription during development and in the adult. We proposed that

conserved subdomains of HSS1 (i.e. Area I, II, and III) contained transcription factor

binding sites that were critical in this process, like the HNF3β element (41). A

comprehensive series of block mutants within the conserved sequences of Area I were

prepared in order to test this hypothesis. Our results indicate that HNF1α and PDX-1

itself are key positive regulators of Area I activation in β cells. These studies suggest that

pdx-1 transcription is regulated by factors associated with controlling both the

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developmental and metabolic states of the β cell, and that an inactivating mutation in one

of its critical regulators (e.g. PDX-1, HNF1α, HNF3β) could affect its expression and

contribute to β cell dysfunction and disease in patients.

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EXPERIMENTAL PROCEDURES

Transfection constructs

Human pdx-1 sequences spanning Area I (-2839 to –2520 bp) were generated by

the polymerase chain reaction (PCR) and cloned directly upstream of the herpes simplex

virus thymidine kinase (Tk) promoter region in the chloramphenicol acetyltransferase

(CAT) expression vector, pTk(An) (42). Pst-Bst:pTk contains mouse pdx-1 sequences

from –2917 (PstI) to –1918 (BstEII) bp (39). Non-complementary transversional block

and point mutations (G to T; C to A) within the conserved sequences of human Area

I:pTk and Pst-Bst:pTk were generated using the Quik Change mutagenesis kit

(Stratagene); the human Area I oligonucleotides used were: S1 Mutant, –2788

CAGTATCAGCGAGGAAGCGACTGACGTCCTGCTAATA –2752; S2 Mutant, -2777

AGGACTATCAGGACGGAAGTAGCCGCCACGACTTTTTCACTGT –2735; S3

Mutant, –2762 TCCTGCTAATAAACGCAGGGGGCACTGTCCACACTTT –2726; S4

Mutant, -2755 AATAAACGACTTTTTACAGTGAATTCCTTTAATTGGTTTAC –

2715; S5 Mutant, -2743

TTTCACTGTCCACACGGGCCGGTTGGGCACCCTTTTTTGTTTATT –2699; S6

Mutant, -2706 TGTTTATTTATCCATCCTCGAGTAGGTTAAATGGCTCGGGA –

2666; S7 Mutant, -2696

TCCATAAGAGCTGCTTGGCCCGGTACCGGGAAGTTGCTCCC –2656; S8 Mutant,

-2684 GCTGTTAAATGGCTC TTTCCTTTGCTCCCTAATGGC –2649; S9 Mutant, -

2671 TCGGGAAGTTGCTCCCGCCGTTAGCGGTATCGCTGCCGG –2633; S2 7-11

Mutant (S2 7-11 MUT), -2777 AGGACTA

TCAGGACGTCCTGAGCAAAAACGACTTTTTCACTGTCC –2733; S2 to β-

Fibrinogen (S2 to β-FIB), –2773

CTATCAGGACGTTTAGTAATATTTGACAGTTTCACTGTCCACACTTT –2726; S2

to insulin A3 (S2 to A3), –2776

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GGACTATCAGGACTAAGACTCTAATTACCCTTTTTTCACTGTCCAC –2731; S5

8-11 Mutant, –2745

TTTTTCACTGTCCACACTTTCCGGGGTTTACACCTTTTTTGTTTA –2701; S5 to

HNF6, –2742 TTCACTGTCCACACTGATATTGATTTTTACCTTTTTTGTTTAT –

2700; S5 to insulin A3, -2743

TTTCACTGTCCACACTAAGACTCTAATTACCCTTTTTTGTTTATT –2699. The

numbering is relative to the human pdx-1 gene. The oligonucleotides used for

mutagenesis of Pst-Bst:pTk were: S2 7-11 Mutant, -2700

AGGACTATCAGGACGTCCTGAGCAAAAAAGACTTTTTCACTGTCC –2656; S2

to β-Fib, -2696

CTATCAGGACGTTTAGTTTAATATTTGACAGTTTCACTGTCCACAGTAT –2649;

S2 to insulin A3, -2690

GGACTATCAGGACTAAGACTCTAATTACGACTTTTTCACTGTC –2657; S5 nt 8-

11 Mutant, -2668

TTTTTCAGTGTCCACACTATCCGGGGTTTACAGCCGTTTTTGTTT –2624; S5 to

HNF6, -2665 TTCACTGTCCACAGTGATATTGATTTTTAGCCGTTTTTGTTTA –

2623, S5 to insulin A3, -2666

TTTCACTGTCCACAGTAAGACTCTAATTACGCCGTTTTTGTTTAT –2622. The

numbering for the Pst-Bst:pTk mutagenesis is relative to the mouse pdx-1 gene. All

mutated sequences are underlined and each construct was verified by sequencing. The

HNF1α expression plasmid (pBJ5-HNF1α (43)) used in the transfection studies was

kindly provided by Dr. Gerald Crabtree (Stanford University, Palo Alto, CA).

Cell transfections

Monolayer cultures of pancreatic islet β (βTC3, HIT T-15, MIN6) and non-β the

(NIH3T3) cells were maintained as described previously (41). The lipofectamine reagent

(Gibco BRL) was used to transfect 1µg each of pdx-1:pTk and the Rous Sarcoma Virus

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(RSV) enhancer-driven luciferase (LUC) expression plasmid, pRSVLUC (44). Co-

transfection studies in NIH3T3 cells were performed similarly with 1 µg each of pdx-

1:pTk, pRSVLUC, and pBJ5 or pBJ5-HNF1α. Extracts were prepared 40 to 48 h after

transfection and LUC (44) and CAT (45) enzymatic assays performed. pdx-1:CAT

activity was normalized to that of pRSVLUC. Each experiment was carried out at least

three independent times.

Electrophoretic mobility shift assays

Gel shift conditions to detect HNF1α (46), HNF4α (47), and HNF6 (48) binding

were carried out as described. Nuclear extracts were prepared using methods described

previously (49). The TNT coupled reticulocyte lysate system (Promega) was used to in

vitro transcribe and translate HNF1α (pGEM7-HNF1α (Richard O’Brien, Vanderbilt

University)), HNF4α (pMT7-HNF4α (50)), HNF6 (pGEM1-HNF6 (51)), PDX-1

(SKII900 (52)), Pax6 (pKW10-Pax6 (53)), Pax4 (pBluescript KSII(+)-Pax4, Beatriz

Sosa-Pineda, St. Jude’s Childrens Research Hospital, Memphis, TN), Nkx 6.1

(pBluescript SKII(+)-Nkx 6.1 (54)), Nkx 2.2 (PCRII-Nkx 2.2, Pelle Serup, Hagedorn

Research Institute, Gentofte, Denmark), and Hblx9 (HB9C (55)). Approximately 5 µg of

extract protein or 5 µl of in vitro translated protein was used per gel-mobility shift

reaction. Anti-PDX-1 antisera was pre-incubated with extract protein for 20 min at room

temperature prior adding the DNA probe; an anti N-terminal (amino acids 1-75; Chris

Wright, Vanderbilt University) and C-terminal (amino acids 271-283 (10)) antisera to

PDX-1 was used in these assays. The same conditions were used with anti HNF1α

(Santa Cruz Biotechnology) and anti HNF1β (Santa Cruz Biotechnology) antisera. The

double-stranded oligonucleotides used to detect binding were end-labeled with

polynucleotide kinase and (γ-32P) ATP. The samples were electrophoresed on 6%

nondenaturing polyacrylamide gels at 150 V for 2 h under high-ionic strength

polyacrylamide gel electrophoresis conditions (15), before drying and autoradiography.

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The probe and competitor sequences were: human S2, -2763

GTCCTGCTAATAAACGACTTTTT –2741; human S2 BLOCK Mutant, -2763

GGAAGTAGCCGCCCCGACTTTTT –2741; human S2 7-11 Mutant, -2763

GTCCTGAGCAAAAACGACTTTTT –2741; human S2 to β-FIB, -2763

GTTTAGTTAATATTTGACAGTTT –2741; human S4, -2745

TTTTTCACTGTCCACACT –2728; human S5, -2732 ACACTTTAATTGGTTTAC –

2715; human E5 BLOCK Mutant, -2732 ACACGGGCCGGTTGGGCACC –2715;

human S5 8-11 Mutant, -2732 ACACTTTCCGGGGTTTAC –2715; human S7-8, -2686

CTGCTGTTAAATGGCTCGGG –2667; HNF1 site in the mouse β-Fib gene (56), -99

TTTAGTTAATATTTGACAGTT –79; HNF1 site in the human insulin gene (57), -229

CCCTGGTTAAGACTCTAATGACC –207; PDX-1 element (termed A3) in the rat II

insulin gene (12), -212 CCTCTTAAGACTCTAATTACCCT –190; HNF4 element in the

human ApoCIII gene (48), -86 GTCTACTGGAAACGGGTC –69; and the HNF6

element in the human HNF3β gene (58), -140 CGATATTGATTTTT –127. The mutated

nucleotides are underlined.

Chromatin immunoprecipitation (ChIP) assays

Monolayer cultures of mouse βTC-3 cells (~0.5-1.0 x 108) were exposed to 1%

formaldehyde in DMEM for five minutes at 23°C; glycine was added to 0.125 M and the

cultures were incubated two minutes. Cells were collected in cold phosphate buffered

saline, pelleted by centrifugation and incubated for 10 min on ice in 0.6 ml of SDS lysis

buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), 1mM PMSF). Lysed

samples were transferred to prechilled microcentrifuge tubes containing 250 mg glass

beads (• 106 µm diameter) and the chromatin was sonicated at power setting 4 with a

Virsonic 100 sonicator (Virtis Company, Inc.) for twelve 10 second pulses at 4°C. The

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reactions were centrifuged for 10 min at 4°C to remove debris and stored at -70°C. A

100 µl aliquot was diluted with 0.9 ml of buffer (0.01% SDS, 1.1% Triton X-100, 1.2

mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, 1mM PMSF, 0.1% protease

inhibitor cocktail for mammalian cells (Sigma)) and precleared with 60 µl of BSA-

blocked protein A-Sepharose for 1h at 4°C. After removal of the Sepharose beads by

centrifugation, 1 µl of anti-PDX-1 antisera, 10 µg of normal rabbit IgG (Santa Cruz

Biotechnology, Inc), or no antibody was added to the supernatant and the reaction was

incubated for 1h at 4°C. Antisera raised to the N- (amino acids 1-75) and C-terminal

(amino acids 271-283 (10)) regions of PDX-1 were used. Antibody-protein-DNA

complexes were isolated by incubation with 60 µl of blocked protein A-Sepharose for 3 h

at 4°C. After extensive washing, bound DNA fragments were eluted and analyzed by

PCR using Ready-to-Go PCR beads (Amersham Pharmacia Biotech, Inc.), 15 pmol of

each primer, and 10 µl of immunoprecipitated DNA per reaction. Cycling parameters

were: 1 cycle of 95°C/2 min and 28 cycles of 95°C/30 sec, 61°C/30 seconds, 72°C/30

sec. The primers used for amplification of mouse Area I were -2785 5’-

CCACTAAGAAGGAAGGCCAG-3’ and -2435 5’-

CTGAGGTTCTTTCTCTGCCTCTCTG-3. Cycling parameters for amplification of

mouse PEPCK were: 1 cycle of 95°C/2 min and 28 cycles of 95°C/30 sec, 61°C/30 sec,

72°C/30 sec. The primers used for amplification of mouse PEPCK were: -434 5’-

GAGTGACACCTCACAGCTGTGG-3’ and -96 5’-

GGCAGGCCTTTGGATCATAGCC-3’. The PCR products were confirmed by

sequencing. Amplified products were electrophoresed through a 1.4% agarose gel in

TAE buffer and visualized by ethidium bromide staining.

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RESULTS

Conserved Area I sequences are essential for β cell-activation.

Transfected pdx-1 driven reporter constructs spanning Area I (-2839 to –2521 bp)

display β cell-specific activation (41). To determine if the conserved sequences between

–2773 to –2643 bp are involved in regulation, each segment (S) of identity was

independently mutated and the effect on Area I activation assayed in transfected HIT-

T15, βTC-3, and MIN6 β cell lines (Figure 1A). Essentially equivalent results were

obtained between these β cell lines (Figure 1B).

Mutations in S4, S5, S6, S7, and S8 all reduced Area I-driven activity (Figure

1B), and their effect was comparable to mutating the HNF3β site (58). The sequences

found within S4 and S7-8 have a nine of thirteen nucleotide match to the consensus

binding site for the HNF4 family of proteins (59-60; Figure 2A). Gel mobility shift

binding assays were performed with a probe corresponding to the HNF4 binding site in

the apolipoprotein CIII gene (apoCIII;(47)), in vitro translated HNF4α, and S4, S7-8, and

apoCIII competitors. In contrast to apoCIII, neither S4 nor S7-8 competed effectively for

HNF4α binding (Figure 2B). There was also no detectable binding between S4 or S7-8

probes and HNF4α (data not shown). These results demonstrated that HNF4α can not

bind to S4 or S7-8, and as a consequence, neither HNF4α nor HNF4γ appear to directly

regulate Area I activation.

HNF1α binds to S2.

The HNF1α and HNF1β homeoproteins bind with identical specificity as homo-

or hetero-dimers (61). S2 has a ten of thirteen nucleotide homology with the consensus

HNF1 binding site (Figure 3A). Binding experiments were performed with the S2 probe,

in vitro translated HNF1α, and nuclear extracts prepared from β (HIT-T15, βTC3), islet

α (αTC6), and pancreatic acinar (AR42J) cell lines and rat liver. The in vitro translated

HNF1α bound complex co-migrated with one formed in liver, acinar, and β extracts

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(Figure 3B). The β cell complex was super-shifted with an antiserum raised to HNF1α,

but not to HNF1β (Figure 3C). However, HNF1β was also found in the HNF1 complex

from kidney extracts (data not shown). These results indicated that HNF1α and HNF1β

could bind to S2. In addition, two other S2 complexes were identified, with the faster

migrating βEF1 (β Enriched Factor 1) complex only detected in β cell extracts and the

other in all (see βEF-1 and U (i.e. for ubiquitous) labeled complexes in Figure 3B).

The binding properties of the S2 complexes formed in β extracts were determined

by competition analyses. All three appeared to be specific, since their formation was

decreased by unlabelled wild-type probe sequences and not by the S2 block mutant

(Figure 3D). In addition, mutating four base pairs within the HNF1α/β binding core

sequence also prevented competition (i.e. S2 7-11 MUT). In contrast, HNF1α binding

was selectively eliminated using either the HNF1 binding site from the β-Fibrinogen gene

(β-FIB) or an S2 mutant that contained the HNF1 binding consensus sequences of the β-

FIB competitor (S2 to β-FIB, 12 of 13 nucleotide homology; Figure 3D).

HNF1α has also been shown to bind in vitro to a distinct control element of the

rat I (28) and human (57) insulin genes. The homology between the human insulin and

the consensus HNF1 binding site is poorer than to S2 (Figure 3A), and competition

analysis demonstrated that HNF1α has a five-fold higher affinity for S2 than the human

site. These results suggest that the diabetic condition found in MODY3 patients may not

only be due to effects on insulin transcription, but also on pdx-1expression.

HNF1α potentiates Area I-driven activation.

To test whether HNF1α regulated Area I activation in β cells, S2 sequences were

changed to resemble the HNF1 binding site of the β-FIB gene. The S2 to β-FIB site

serves as an effective competitor and probe for HNF1α binding, although it has little or

no effect on the other S2 complexes (Figure 3D; data not shown). This altered

specificity mutant was made in Area I alone or Area I/Area II combined. The larger

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mouse pdx-1 gene promoter construct, termed Pst-Bst, directs β cell-selective expression

in transfected β cell lines and transgenic animals (39).

The activity of the S2 to β-FIB mutant in Area I:pTk and Pst-Bst:pTk was

compared to the wild-type and S2 7-11 mutant. We believed that the S2 7-11 core mutant

would be less active than the wild-type, as this mutation prevents protein complex

formation (Figure 3). Each of these plasmids was introduced into HIT-T15, βTC-3, and

MIN6 cells. As expected, the S2 7-11 mutant reduced both Area I and Pst-Bst mediated

activation (Figure 4A,B); the effect was more pronounced in Pst-Bst:pTk (Figure 4B).

The altered specificity mutant was consistently more active than the S2 7-11 mutant in

Pst-Bst, although not in Area I alone. Similarly, the HNF3β binding site mutant in Area II

only decreased β cell stimulation within a Pst-Bst context, while both Area I and Pst-Bst

activation were decreased by the HNF3β binding mutant in Area I (41).

To directly determine if HNF1α activated Area I expression, an HNF1α

expression plasmid was co-transfected with either the wildtype or S2 block mutant Area

I:pTk construct into NIH 3T3 cells, which lack endogenous HNF1α. Overexpression of

HNF1α activated Area I:pTk but had little effect on the S2 block mutant (Figure 4C).

Collectively, these results suggest that HNF1α regulates pdx-1 activation, but like

HNF3β stimulation of Area II, this process involves functional interactions with a β cell

activator(s) residing in a neighboring control region.

HNF6 binds inefficiently to S5.

Although S5 is highly homologous to the consensus HNF6 binding site, in vitro

translated HNF6 bound very poorly when compared to the HNF6 site of the HNF3β

promoter (Figure 5A,B). Similarly, S5 was only a slightly more effective HNF6 binding

competitor than the S5 block mutant (Figure 5C). In addition, HNF6 binding to S5 was

not detected in β or non-β (BHK and liver) nuclear extracts, although binding was readily

observed with the HNF3β site probe (Figure 5D).

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The prominent S5 binding complex was unique to β cell lines, and termed βEF2

(Figure 5D). Competition analysis demonstrated that βEF2 binding was reduced by the

S5 wild-type competitor, but not the S5 block mutant or HNF3β promoter competitor

(Figure 5E). These results suggested that the protein(s) that forms the βEF2 complex is

the S5 activator, and not HNF6. The inability to see a change in PDX-1 protein levels in

HNF6 null mice also indicates that this factor does not regulate pdx-1 transcription (35).

βEF1 and βEF2 contain PDX-1.

The data from the preceding experiments indicated that a β cell-enriched

protein(s) binds to S2 and S5. Because these segments contain the characteristic

homeodomain core binding sequence (i.e. TAATA), the islet-enriched PDX-1, Pax4,

Pax6, Nkx2.2, Nkx6.1, and Hlxb9 homeodomain proteins were analyzed for binding to

S2 (Figure 6A) and S5 (Figure 6B). Binding was only observed with in vitro translated

PDX-1, and the complex formed co-migrated with βEF1 and βEF2 (Figure 6 A and B).

Pre-incubating the β extract with anti-PDX-1 antisera also specifically eliminated or

super-shifted the βEF-1 (Figure 6C) and βEF-2 (Figure 6D) complexes. As a final test for

PDX-1binding, a competition experiment was performed with the PDX-1 binding A3

element of the insulin gene. As expected, this competitor prevented formation of βEF-1

(Figure 6C) and βEF-2 (Figure 6D), although it did not affect HNF1α binding to S2

(Figure 6C). S5 and A3 were found to bind with similar efficacy to PDX-1 in these

assays, and 5-fold higher than S2 (data not shown). These data demonstrated that PDX-1

can bind in vitro to two distinct cis-acting regulatory elements of Area I.

PDX-1 stimulates Area I activation.

To determine if PDX-1 binding to Area I mediated stimulation of Area I:pTk and

Pst-Bst:pTk, S2 and S5 sequences were changed to more closely resemble the insulin A3

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element. The S2 mutation specifically eliminated HNF1α binding (Figure 6C; data not

shown). The activity of the A3 conversion mutants was compared in β cells to the

wildtype and the core element block mutant.

There was little or no difference on S2 activation of Area I alone in the A3

conversion mutant versus the 7-11 mutant in HIT-T15 and βTC3 cells, although

stimulation was observed in MIN6 cells (Figure 7A). In contrast, Pst-Bst activity was

stimulated in all of the β cell lines by the S2 to A3 mutation (Figure 7B). The A3

conversion mutant in S5 more profoundly influenced activation, as both Area I (Figure

7C) and Pst-Bst (Figure 7D) activities were increased. Depending upon the cell line,

Area I alone activation was potentiated 6- to 13-fold over the core mutant, and 1.5- to

2.5-fold over the wild-type. The S5 to A3 conversion mutation in Pst-Bst was

essentially the same as wild-type Pst-Bst. These results suggested that binding of PDX-1

to S5 and/or S2 was involved in regulating pdx-1 transcription.

PDX-1 binds to Area I in vivo.

The chromatin immunoprecipitation (ChIP) assay is a powerful tool for analyzing

the occupancy of transcription factors on their cognate binding elements in vivo (62-63).

In order to more fully address the possibility of PDX-1 mediated regulation of pdx-1 gene

expression, we used the ChIP assay to determine if PDX-1 binding to Area I could be

observed within the context of the endogenous gene. Immunoprecipitation of

formaldehyde cross-linked chromatin from βTC3 cells with antibodies specific to the N-

or C-terminal region of PDX-1 precipitated Area I sequences, whereas rabbit IgG, CDX-4

specific antisera, or the no antibody controls did not (Figure 8; top panel). In contrast,

anti-PDX-1 antisera did not immunoprecipitate promoter sequences from the

phosphoenolpyruvate kinase (PEPCK) gene, which is not transcribed in βTC3 cells

(Figure 8; bottom panel). Similar results were observed with MIN6 cells (data not

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shown). Together with the gel shift and transfection data described above, these results

demonstrate that PDX-1 regulates its own transcription.

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DISCUSSION

PDX-1 plays a critical role in both pancreatic development and adult islet β cell

function. The nuclease HSS1 region (-2560 to -1880 bp) of the pdx-1 gene contains

crucial cis-acting elements involved in expression. The basis for this proposal was

supported by data demonstrating that: (1) only HSS1 was independently capable of

directing islet β cell selectively expression in transfection assays performed with pdx-1

driven constructs spanning the 4.5 kb promoter region (39); (2) the only significant

sequence conservation within the promoter region was found within HSS1, as defined by

the distinct functional subdomains (i.e. Areas I, II, and III) (41); and (3) a pdx-1 driven

transgenic construct spanning Areas I and II was selectively expressed in islet β cells in

mice (39). Collectively, these results strongly suggested that HSS1 was regulated by

transcription factors critical for β cell formation and function. To localize regulatory

elements shared amongst the mammalian pdx-1 genes, the segments of identity within

Area I were specifically mutated and the effect on activation in β cells determined in

transfection assays. The analyses performed in HIT-T15, βTC-3, and MIN6 cells

localized sequences that were involved in stimulation by this HSS1 subdomain (S2, S4,

S5, S6, S7, S8), as well as conserved segments of little or no regulatory importance (S1,

S3, S9). In addition, HNF-1α and PDX-1 itself were shown to mediate activation from

Area I.

Strikingly, a transcription factor database analysis of the mutational sensitive

sequences in Area I suggested that proteins critical in endocrine cell development (HNF6:

S5) and function (HNF1: S2; HNF4: S4, S7-8) contribute to activation. However, only

HNF1α was found to bind segment specific probes in gel shift experiments performed

with in vitro translated HNF1α, HNF4α, and HNF6 or nuclear extracts prepared from

factor producing cells. Our inability to detect HNF4α or HNF6 binding strongly suggests

that these key regulatory proteins do not directly control Area I activation. The absence of

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a change in PDX-1 protein levels in HNF6 null mice also indicates that this factor does

not play any role in regulating pdx-1 transcription (35). Endocrine islet cell development

was severely impaired in these mice, apparently due to the requirement for HNF6 in

neurogenin 3 expression (35), a transcription factor that is critical in determination of

endocrine islet precursors (64).

To test if HNF1α regulated Area I activation in β cells, S2 sequences were

changed to select for HNF1α binding (S2 to β-FIB) within the context of pdx-1 reporter

constructs driven by human Area I alone or mouse Pst-Bst, which spans Areas I and II.

The activity of the altered specificity mutants were compared to the wildtype as well as

the HNF1α binding defective mutant construct, S2 7-11 (Figure 4). The S2 to β-FIB

mutant only effectively activated the pdx-1 construct spanning Areas I and II suggesting

that stimulation by HNF1α involves functional interactions with an Area II activator, a

situation that parallels the requirement of Area I for HNF3β activation of Area II (41).

HNF1α was also shown to specifically activate S2 directed expression in cotransfection

assays in NIH 3T3 cells. In contrast to S2, the HNF1α binding site recently described in

the human pdx-1 gene may not be of general regulatory significance, as it is not

conserved within the mouse or chicken genes (64).

The HNF-1 family of transcription factors bind DNA as homo- or heterodimers

(61). Interestingly, HNF1β is co-expressed with pdx-1 in the ventral and dorsal walls of

the primitive foregut during early pancreas development (66). In contrast, HNF1α is

expressed at a later developmental stage and at much higher levels than HNF1β in adult β

cells (66). We were only able to detect HNF1α in the S2 specific HNF1 complex from

HIT-T15, βTC-3, and MIN6 nuclear extracts, although HNF1β was independently shown

to be capable of binding to S2 (data not shown). Thus, these results support the

possibility that HNF1α and/or HNF1β function as activators of pdx-1 expression during

pancreas specification and in the adult islet β cell.

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The decrease in insulin synthesis and secretion found in mice lacking HNF1α also

provides support for a role in pdx-1 transcription (30,67), as decreased expression of

PDX-1 would be expected to have a debilitating effect upon β cell function, through its

actions on GLUT2 and insulin transcription. Two distinct lines of HNF1α null mice have

been independently derived by Lee et. al. (30) and Pontoglio et. al. (68). The same

HNF1α sequences were targeted for removal (i.e. amino acids 1-108), although each used

a different strategy to eliminate them. Both lines result in decreased insulin mRNA

expression. However, the absence of HNF1α expression had a distinct effect on islet

pdx-1 expression as well as the overall physiology of these animals. The decrease in pdx-

1 mRNA expression in HNF1α null mice from Pontoglio et al. was 2.4 -fold in the

newborn pancreas islets and 2.9-fold in the adult (Figure 1A, Reference 69), whereas

little of no effect was found in those from Lee et. al. in islets from two week old mice

(Figure 3, Reference 70). It is unclear which of these mouse models more closely

resembles regulation in the human.

In addition to HNF1α, PDX-1 was found to specifically bind to S2 in gel shift

assays performed with β-cell nuclear extracts (Figure 6A and C) and was the principal S5

binding activity detected in these extracts (Figure 6B and D). Furthermore, Area I and

Pst-Bst mediated activation were compromised in the S5 8-11 mutant (Figure 7C and D),

demonstrating the critical nature of this site in transcriptional regulation. PDX-1 activated

the altered specificity mutants that replaced S2 or S5 with insulin A3. Importantly, PDX-

1 was shown to specifically bind to Area I of the endogenous pdx-1 gene using the ChIP

assay. In contrast, we were unable to demonstrate HNF1α binding to Area I in vivo (data

not shown), which may either reflect occupancy of S2 by PDX-1 or simply a technical

inability to recover HNF1α bound complexes in our ChIP assay.

These data strongly suggest that pdx-1 transcription is autoregulated. However,

PDX-1 is not absolutely required for transcription as expression is detected during

embryogenesis in pdx-1-/- mice (9), and in the presence of a dominant negative PDX-1

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mutant in βTC-3 cells (71). Autoregulation is utilized by other mammalian

homeodomain-encoding genes, apparently to prevent extreme fluctuations in expression

(72-77). Because PDX-1 is the major S5 binding factor, it is likely to bind and regulate

through this site. Support for this proposal is also provided by quantitative competition

assays showing that PDX-1 binds with greater affinity to S5 than S2 in vitro, and

transfection data indicating that PDX-1 can transactivate through S5 (78). We are

currently working towards developing model systems to address the importance of PDX-

1 autoregulation in vivo.

Islet β cell-specific expression clearly relies on the activity of many cell-enriched

transcription factors, which function cooperatively to impart control (1,3). The data

presented have defined conserved cis-acting regulatory elements that are necessary for

pdx-1 expression in the β cell. Furthermore, it is possible that the Area I sites that were

insensitive to mutation in our assays reflect the limitations of our tumor cell lines, rather

than their importance in regulation in vivo. Identifying the regulatory factors that control

the expression of genes such as pdx-1 may provide insight into the inherited defects that

cause insulin deficiency and diabetes. For example, defects in β-cell function in MODY

patients expressing a dysfunctional HNF1α or PDX-1 protein may in part result from

defects in pdx-1 expression. As reduced PDX-1 levels would, in turn, likely have

profound consequences on expression on many of the target genes involved in glucose

sensing, including insulin, glucokinase, and GLUT2.

ACKNOWLEDGEMENTS

We are grateful to Drs. Chris Wright, and Maureen Gannon for assistance in

designing and interpreting many of the experiments described here. This work was

supported by grants from the National Institutes of Health (NIH RO1 DK50203 to R.S.)

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and partial support from the Vanderbilt University Diabetes Research and Training

Center Molecular Biology Core Laboratory (Public Health Service grant P60 DK20593

from the National Institutes of Health).

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FIGURE LEGENDS

Figure 1. Localization of transcriptional control segments within conserved Area I

sequences. A. Conserved sequences within Area I sequences are shaded (41). The

nucleotides are numbered relative to the human pdx-1 gene. Each bar spans the mutated

sequences; the HNF3β control element is labeled. B. Wild-type and mutant Area I:pTk

CAT were transfected into the HIT-T15, βTC3, and MIN6 cell lines. The percent mutant

Area I:pTk activity + standard deviation (SD) from at least 3-5 independent experiments

is presented relative to wild-type activity. Not determined (ND).

Figure 2. HNF4α does not bind to S4 or S7-8. A. The homology between S4, S7-8,

and the HNF4 site in the human apolipoprotein CIII (apoCIII) (47) to the consensus

HNF4 binding site (74) (RGKNYRRRGKYYN, where R=purine, G or A; K=G or T;

N=A, C, G, or T; Y=pyrimidine, C or T) is shown. Non-matching nucleotides are in

lowercase. B. Gel mobility shift assays were performed with the apoCIII probe in the

presence of in vitro translated HNF4α and the S4, S7-8, apoCIII competitors. The

competitions were performed in the absence (-) or presence of 50, 100, or 200 fold molar

excess of unlabelled competitor to probe.

Figure 3. Binding of liver and β cell nuclear proteins to S2.

A. The homology between the consensus HNF1 binding site (61), the β-

fibrinogen (β-FIB) HNF1 site, and wild-type and mutant S2 sequences is illustrated

(GTTAATNATTRRC; N= A, C, G, or T and R= purine, G or A). Non-matching

nucleotides are in lowercase.

B. In vitro translated (IVT) HNF1α (lane 1) and equal concentrations of liver

(lane 2), αTC-6 (lane 3), AR42J (lane 4), HIT-T15 (lane 5), or βTC-3 (lane 6) nuclear

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protein extract were analyzed for S2 binding. The arrows designate a specific binding

complex; HNF1α ,βEF1, and the U complexes discussed in the text are labeled.

C. The HNF1α and HNF1β antisera were preincubated with HIT-T15 nuclear

extract before initiation of the S2 DNA-binding reaction. The position of the HNF1α and

super-shifted (SS) HNF1α complexes are shown. Lane 1 represents the binding reaction

conducted in the absence of antisera (-).

D. S2 binding reactions were conducted with HIT-T15 nuclear extract either

alone (lane 1) or in the presence of a 200-fold molar excess of S2 (lane 2), S2 Block

MUT (lane 3), S2 7-11 MUT (lane 4), S2 to β-FIB (lane 5), or β-FIB (lanes 6) competitor

to the S2 probe.

Figure 4. HNF1α stimulates Area I-driven expression in β cells.

A and B. The pdx-1 Area I (A) and Pst-Bst (B) region reporter plasmids are

shown diagrammatically. Area I is shaded in the S2 core block mutant (S2 7-11) and

hatched in the S2 altered specificity mutant (S2 to β-FIB). The wild-type and mutant

Area I:pTk (A) and Pst-Bst:pTk (B) plasmids were transfected into HIT-T15, βTC3, and

MIN6 cell lines. The normalized activity ± S.D. of each Area I mutant construct is

presented as the percent activity of the corresponding wild-type plasmid.

C. NIH 3T3 cells were co-transfected with an HNF1α expression vector (pBJ5-

HNF1α or vector alone (pBJ5) and the wildtype or S2 block mutant Area I:pTk. Area I is

shaded in the S2 block mutant (S2 BLOCK MUT). The normalized activity + S.D. of

each construct is presented as fold pBJ5-HNF1α activation relative to pBJ5.

Figure 5. S5 binds a β cell-enriched factor, but not HNF6.

A. Sequences of S5, the S5 core block mutant (S5 8-11 MUT) and the HNF6

binding site from the HNF3β promoter are shown. The nucleotide similarity to the

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consensus HNF6 binding site is also shown, with non-matching nucleotides in lower case

(57); D=A, T, or G; H=A, T, or C; W=A or T; Y= pyrimidine, T or C).

B. S5 and the HNF3β promoter probes were incubated with in vitro translated

(IVT) HNF6. The binding reactions with S5 and HNF3β promoter probes contained 5

and 1 µl of the HNF6 translation reaction, respectively. The HNF6 complex is indicated

with an arrow.

C. Binding reactions were conducted with the HNF3β promoter probe using liver

nuclear extracts. A 100-fold molar excess of unlabeled S5, S5 8-11 MUT, and HNF3β

promoter competitor to probe was used.

D. Nuclear extracts from liver, BHK, HIT-T15, βTC3, or MIN6 cells were

analyzed for S5 (Lanes 1-5) and HNF3β promoter (Lanes 6-10) binding. The principal

complex formed with the S5 probe in liver extracts is non-specific (NS) (data not shown).

Gel shift assays performed with HNF6 specific antiserum confirmed the presence of

HNF6 in the complex detected specifically with the HNF3β probe in liver and β nuclear

extracts (data not shown)).

E. Binding reactions were conducted with the S5 probe and βTC-3 extracts either

alone (-) or in the presence of a 100-fold molar excess of S5, S5 8-11 MUT, or the

HNF3β promoter competitor to probe. Note that only S5 competed effectively with the

probe for βEF2 binding.

Figure 6. PDX-1 is present in the βEF1 and βEF2 complexes.

A. and B. Gel mobility shift assays with the S2 (A) and S5 (B) probes were

performed in absence (-) and presence of in vitro translated PDX-1, Pax4, Pax6, Nkx2.2,

Nkx6.1, or Hlxb9 homeodomain protein. Translation reactions conducted with 35S-

methionine demonstrated that a similar amount of protein was present in each reaction

(data not shown). The binding complexes formed with HIT-T15 nuclear extract (β NE)

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are shown. The HNF1α and βEF binding complexes described in the text are labeled, as

are the non-specific (NS).

C. and D. HIT-T15 nuclear extract binding to the S2 (C) and S5 (D) probes was

conducted in absence (-) or presence (+) of N- or C-terminal PDX-1 antisera, and a 200-

fold molar excess of the S2, S5, or insulin A3 competitor.

Figure 7. PDX-1 stimulates Area I-driven expression in β cells.

Area I (A,C) and Pst-Bst (B,D) region reporter plasmids are shown

diagrammatically. Area I is shaded in the core block mutant in S2 (S2 7-11) and S5 (S5

8-11) and hatched in the A3 conversion mutation. The wild-type and mutant Area I:pTk

and Pst-Bst:pTk plasmids were transfected into HIT-T15, βTC3, and MIN6 cell lines.

The normalized activity ± S.D. of each Area I mutant construct is presented as the percent

activity of the corresponding wild-type plasmid. (A,B) S2; (C,D) S5.

Figure 8. PDX-1 occupies Area I in vivo.

Cross-linked chromatin from βTC3 cells was incubated with antibodies raised to

the N- (Lane 3) or C- (Lane 4) terminal region of PDX-1. The immunoprecipitated DNA

was analyzed by PCR for Area I (top panel) and PEPCK (bottom panel) sequences. As

controls, PCR reactions were run with no DNA (Lane 1), total input chromatin (Lane 2),

and DNA obtained from immunoprecipitation with an antibody raised to CDX-4 (Lane

5), rabbit IgG (Lane 6) or no antibody (Lane 7). These experiments were carried out 9

independent occasions.

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apoCIIIS4 WT S7-8 WT

S4 WT AaGTgAcAGGTgT 9/13

S7-8 WT GtTAaAtGGcTCG 9/13

apoCIII tGGGCAAAGGTCA 12/13

CONSENSUS RGKNYRRRGKYYN

HOMOLOGYA.

B.

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HNF1α

βEF1

S2 WT GcTAATAAacGAC 10/13

S2 BLOCK MUT tagccgcccCGAC 4/13

S2 nt 7-11 MUT GagcAaAAACGAC 9/13

β-FIB GTTAATAtTTGAC 12/13

INS HNF1 GTTAAgAcTctAa 8/13

CONSENSUS HNF1 GTTAATNATTRRC

HOMOLOGY

A.

B.

U

IVT HNF1

α

LIVER

αTC6

AR42J

HIT-T15

βTC3

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SS

- anti-HNF1α

anti-HNF1β

C.

HNF1α

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HNF1α

-

D.

COMPETITOR:

U

S2 S2 to

β-FIB MUT

S2 BLOCK MUT

S2 7-11 MUT

β-FIB

βEF1

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FOLD ACTIVATION

0 4 2 6 8 10

6.7

1.4

Area I I

S2 BLOCK MUT I

C.

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CONSENSUS HNF6

TTAATTGgTTTAc

GATATTGATTTTT

TTccggGgTTTAc

DHWATTGAYTWWD

S5 11/13

HNF3β 13/13

7/13S5 8-11 MUT

HOMOLOGY

A.

S5 HNF3

βProbe:

HNF6

IVT HNF6 (µl) 5 1

B.

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- S5 S5 8-11 MUT

COMPETITOR:

C.

HNF6

HNF3

β

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PROBE

D.

S5 HNF3β

HNF6NS

βEF2

LIVER

βTC3

HIT-T15

MIN6

BHK

LIVER

βTC3

HIT-T15

MIN6

BHK

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-

βEF2

E.

COMPETITOR: S5 S5 8-11 MUT

HNF3

β

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A.

U

HNF6

HNF1

βNE

PDX-1

PAX4

PAX6

NKX2.2

NKX6.1

Hblx9

βEF1

NSNS

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bEF2

B.

βEF2

βNE

PDX-1

PAX4

PAX6

NKX2.2

NKX6.1

Hblx9

NSNS

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+-- +

N-TermC-term[αPDX-1

C.

+-- +

S2Insulin A3[Competitor

HNF1α

U

βEF1

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+-- +

N-TermC-term[αPDX-1

D

+-- +

S5Insulin A3[Competitor

SS

βEF2

.

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rabbit IgG

No Ab

Area I

PEPCK

12

34

56

7

No DNA

Input (1/100 dil)

αPDX-1 (N-term)

αPDX-1 (C-term)

αcdx4

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Kevin E. Gerrish, Michelle A. Cissell and Roland Steinpdx-1 gene

The role of hepatic nuclear factor 1a and PDX-1 in transcriptional regulation of the

published online October 5, 2001J. Biol. Chem. 

  10.1074/jbc.M109244200Access the most updated version of this article at doi:

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