2316?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 6? ? ? June 2008
Development of type 2 diabetes
following intrauterine growth retardation
in rats is associated with progressive
epigenetic silencing of Pdx1
Jun H. Park,1 Doris A. Stoffers,2,3 Robert D. Nicholls,4 and Rebecca A. Simmons1,3
1Department of Pediatrics, Children’s Hospital of Philadelphia, 2Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, and
3Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
4Birth Defects Laboratories, Division of Medical Genetics, Department of Pediatrics, Children’s Hospital of Pittsburgh, and
Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Intrauterine growth retardation (IUGR), a common complication
of pregnancy, has been linked to the later development of diseases
in adulthood such as type 2 diabetes (1). It has been hypothesized
that the molecular mechanisms underlying this phenomenon
may in part be related to epigenetic modulation of expression of
key developmental genes (2, 3). Epigenetic modifications provide
a mechanism that allows the stable propagation of gene activity
states from 1 generation of cells to the next. Epigenetic states can
be modified by environmental factors, which may contribute to the
development of abnormal phenotypes. In mammals, DNA meth-
ylation and histone modifications represent the major epigenetic
mechanisms implicated in the regulation of gene transcription.
We have developed an animal model of IUGR caused by utero-
placental insufficiency, which limits the supply of critical sub-
strates and hormones to the fetus (4, 5). This abnormal metabolic
intrauterine milieu affects the development of the fetus by per-
manently modifying gene expression and function of susceptible
cells, such as the β cell (4–6), and leads to the development of dia-
betes in adulthood (4–6).
Pdx1 is a pancreatic and duodenal homeobox 1 transcription
factor that regulates pancreas development and β cell differentia-
tion. Both genetic and acquired reductions in Pdx1 expression in
humans and in animal models have been shown to cause type 2 dia-
betes, β cell dysfunction (7–14), and impaired islet compensation
in the presence of insulin resistance (7, 8). In IUGR rats, we have
observed that Pdx1 mRNA levels are reduced by more than 50% in
IUGR fetuses (6). At birth, β cell mass is normal but Pdx1 mRNA
levels are decreased in IUGR rats (6). In IUGR adult rats, β cell mass
is markedly decreased and Pdx1 expression is nearly absent (6).
IUGR leads to the permanent suppression of Pdx1 in islets,
suggesting that an epigenetic mechanism may be responsible via
changes in DNA methylation, histone modifications, or chroma-
tin protein binding (15). The region spanning the proximal 5ʹ pro-
moter and all of exon 1 of Pdx1 contains a highly conserved CpG
island. Deletion of the proximal promoter completely abolishes
Pdx1 promoter activity (16). This region is also heavily acetylated
at histone H3 and H4 in a β cell tumor line (17). However, the epi-
genetic characteristics of the Pdx1 gene have not been examined in
primary islet tissue in normal or disease states.
In the present study, we hypothesized that epigenetic changes involv-
ing histone modifications, DNA methylation, chromatin remodeling,
and transcription were responsible for Pdx1 silencing during the tran-
sition from IUGR to diabetes in adults. We determined that in early
postnatal life, prior to the onset of diabetes, the IUGR state induces
deacetylation of histones H3 and H4, which is facilitated by recruit-
ment of histone deacetylase 1 (HDAC1) and Sin3A to the proximal
Nonstandard?abbreviations?used: 5-AzaC, 5-aza-2ʹ-deoxycytidine; Dnmt, DNA
methyltransferase; HDAC, histone deacetylase; H3K4, histone 3 lysine 4; H3K9,
histone 3 lysine 9; H3K4me3, trimethylation of lysine 4 at H3; H3K9me2, dimethyl-
ation of lysine 9 at H3; IUGR, intrauterine growth retardation; TSA, trichostatin A.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 118:2316–2324 (2008). doi:10.1172/JCI33655.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 6 June 2008
promoter of Pdx1. Loss of acetylation is accompanied by loss of bind-
ing of the key transcription factor, USF-1. As the disease state pro-
gresses, histone 3 lysine 4 (H3K4) is demethylated and histone 3 lysine
9 (H3K9) is methylated. Finally, once diabetes occurs, methylation of
the CpG island in the proximal promoter of Pdx1 ensues, locking in
marked suppression of Pdx1. These are the first studies to our knowl-
edge to describe the ontogeny of chromatin remodeling in vivo from
the fetus to the onset of a disease in adulthood.
Pdx1 transcription is reduced in IUGR islets. In previous studies, we
found that Pdx1 mRNA levels were decreased by 50% in fetuses
and 2-week-old pups and 80% in adult IUGR animals (6). To
determine whether this reduction in Pdx1 mRNA levels was sec-
ondary to a reduction in transcription of the Pdx1 gene or to an
increase in mRNA stability, we induced IUGR in Pdx1-LacZ in
vivo transcriptional reporter mice. The transgene was regulated
by the 4.6-kb XbaI-XbaI fragment containing 4.5 kb of the mouse
Pdx1 promoter and most of the 5ʹ untranslated region (UTR).
Most of the important Pdx1 enhancers described to date are
included within this promoter fragment (17–19). Endogenous
Pdx1 mRNA levels are reduced by 50.4% and transgene LacZ
mRNA is reduced in parallel by approximately 57.1% (P < 0.05
versus control). This indicates that IUGR regulates Pdx1 mRNA
by reducing the activity of transcriptional promoter or enhancer
elements in the proximal 4.5-kb Pdx1 5ʹ region.
Methylation status of the Pdx1 gene promoter in IUGR and control islets.
The proximal 5ʹ flanking region of the Pdx1 gene and its first exon
include a highly conserved CpG island that encompasses nucleo-
tides –360 to +200 relative to the translational start site (Figure
1A). Active promoters are associated with unmethylated CpG
islands and open chromatin structure, whereas inactive promot-
ers are typically characterized by a repressed chromatin structure
and hypermethylated CpGs. We determined the DNA methylation
Methylation status of proximal promoter of Pdx1. (A) Map of Pdx1.
The proximal promoter of Pdx1 is part of a more extensive CpG island.
Filled circles represent CpG dinucleotides, and numbers indicate posi-
tions relative to the transcriptional start site. The position of the E-box
bound by USF-1 is indicated. (B) Methylation of CpG dinucleotides
within the promoter of Pdx1. Islets were isolated from 5 IUGR and 5
control animals at 6 months of age, and Pdx1 DNA methylation was
quantified by pyrosequencing of bisulfite-treated DNA. We did not
detect methylation at any CpG of Pdx1 in controls. The shaded bars
represent data obtained and averaged from 5 IUGR animals and pre-
sented as percent methylation at each CpG site. Data are ± SEM.
Quantitative analysis of Dnmt1, Dnmt3a, and Dnmt3b
bound at the Pdx1 promoter region. ChIP analysis of
cross-linked chromatin from islets of IUGR and control
animals at 2 weeks and 6 months of age IP with anti-
body to Dnmt1, Dnmt3a, and Dnmt3b. Control (Con) and
IUGR samples were run on the same gel. Input DNA (In)
represents PCR products without prior IP. The IgG IP
showed negligible PCR product, indicating little or no IP
in the absence of primary antibody. The relative amount
of Dnmt1-, Dnmt3a-, and Dnmt3b-bound Pdx1 promoter
was measured by genomic quantitative real-time PCR
(Q-PCR) and normalized to input DNA. Data are repre-
sented as percent of control values. n = 3 experiments,
data are ± SEM; *P < 0.05 versus controls.
2324?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 6 June 2008
and Harrison, L.C. 1994. Insulin-promoter-factor
1 is required for pancreas development in mice.
11. Offield, M.F., et al. 1996. PDX-1 is required for pan-
creatic outgrowth and differentiation of the rostral
duodenum. Development. 122:983–995.
12. Hui, H., and Perfetti, R. 2002. Pancreas duodenum
homeobox-1 regulates pancreas development during
embryogenesis and islet cell function in adulthood.
Eur. J. Endocrinol. 146:129–141.
13. Ahlgren, U., Jonsson, J., Jonsson, L., Simu, K., and
Edlund, H. 1998. Beta-cell-specific inactivation of
the mouse Ipf1/Pdx1 gene results in loss of the
beta-cell phenotype and maturity onset diabetes.
Genes Dev. 12:1763–1768.
14. Stoffers, D.A., Zinkin, N.T., Stanojevic, V., Clarke,
W.L., and Habener, J.F. 1997. Pancreatic agenesis
attributable to a single nucleotide deletion in the
human IPF1 gene coding sequence. Nat. Genet.
15. Jaskelioff, M., and Peterson, C.L. 2003. Chromatin
and transcription: histones continue to make their
marks. Nat. Cell Biol. 5:395–399.
16. Sharma, S., et al. 1996. Pancreatic islet expression of
the homeobox factor STF-1 relies on an E-box motif
that binds USF. J. Biol. Chem. 271:2294–2299.
17. Gerrish, K., Van Velkinburgh, J.C., and Stein,
R. 2004. Conserved transcriptional regula-
tory domains of the pdx-1 gene. Mol. Endocrinol.
18. Gannon, M., Gamer, L.W., and Wright, C.V. 2001.
Regulatory regions driving developmental and tis-
sue-specific expression of the essential pancreatic
gene pdx1. Dev. Biol. 238:185–201.
19. Stoffers, D.A., Stanojevic, V., and Habener, J.F.
1998. Insulin promoter factor-1 gene mutation
linked to early-onset type 2 diabetes mellitus
directs expression of a dominant negative isopro-
tein. J. Clin. Invest. 102:232–241.
20. Bird, A.P., and Wolffe, A.P. 1999. Methylation-
induced repression-belts, braces, and chromatin.
21. Li, H., et al. 2006. The histone methyltransferase
SETDB1 and the DNA methyltransferase DNMT3A
interact directly and localize to promoters silenced
in cancer cells. J. Biol. Chem. 281:19489–19500.
22. Liang, G., et al. 2004. Distinct localization of
histone H3 acetylation and H3-K4 methylation to
the transcription start sites in the human genome.
Proc. Natl. Acad. Sci. U. S. A. 101:7357–7362.
23. Nightingale, K.P., et al. 2007. Cross talk between
histone modifications in response to HDAC
inhibitors: MLL4 links histone H3 acetylation
and histone H3K4 methylation. J. Biol. Chem.
24. Huang, Y., Fang, J., Bedford, M.T., Zhang, Y., and
Xu, R.M. 2006. Recognition of histone H3 lysine-4
methylation by the double tudor domain of JMJD2A.
25. Qian, J., Kaytor, E.N., Towle, H.C., and Olson, L.K.
1999. Upstream stimulatory factor regulates Pdx-1
gene expression in differentiated pancreatic β-cells.
Biochem. J. 341:315–322.
26. Sharma, S., et al. 1996. Pancreatic islet expres-
sion of the homeobox factor STF-1 (Pdx-1) relies
on an E-box motif that binds USF. J. Biol. Chem.
27. Scaglia, L., Cahill, C.J., Finegood, D.T., and Bonner-
Weir, S. 1997. Apoptosis participates in the remod-
eling of the endocrine pancreas in the neonatal rat.
28. Del Guerra, S., et al. 2005. Functional and molecu-
lar defects of pancreatic islets in human type 2 dia-
betes. Diabetes. 54:727–735.
29. Plaisance, V., et al. 2006. MicroRNA-9 controls the
expression of Granuphilin/Slp4 and the secretory
response of insulin-producing cells. J. Biol. Chem.
30. Haigis, M.C., et al. 2006. SIRT4 inhibits gluta-
mate dehydrogenase and opposes the effects of
calorie restriction in pancreatic beta cells. Cell.
31. Shi, Y., and Whetstine, J.R. 2007. Dynamic regula-
tion of histone lysine methylation by demethylases.
Mol. Cell. 25:1–14.
32. Forneris, F., et al. 2006. A highly specific mechanism
of histone H3-K4 recognition by histone demethyl-
ase LSD1. J. Biol. Chem. 281:35289–35295.
33. Klose, R.J., et al. 2007. The retinoblastoma bind-
ing protein RBP2 is an H3K4 demethylase. Cell.
34. Trewick, S.C., Henshaw, T.F., Hausinger, R.P., Lin-
dahl, T., and Sedgwick, B. 2002. Oxidative demeth-
ylation by Escherichia coli AlkB directly reverts
DNA base damage. Nature. 419:174–178.
35. Cloos, P.A., et al. 2006. The putative oncogene
GASC1 demethylates tri- and dimethylated lysine
9 on histone H3. Nature. 442:307–311.
36. Litt, M.D., Simpson, M., Gaszner, M., Allis, C.D.,
and Felsenfeld, G. 2001. Correlation between
histone lysine methylation and developmental
changes at the chicken beta-globin locus. Science.
37. Noma, K., and Grewal, S.I. 2002. Histone H3 lysine
4 methylation is mediated by Set and promotes
maintenance of active chromatin states in fission
yeast. Proc. Natl. Acad. Sci. U. S. A. 99:16438–16445.
38. He, B., et al. 2003. SOCS-3 is frequently silenced
by hypermethylation and suppresses cell growth
in human lung cancer. Proc. Natl. Acad. Sci. U. S. A.
39. Cerda, S., and Weitzman, S.A. 1997. Influence of
oxygen radical injury on DNA methylation. Mutat.
40. Ihara, Y., et al. 1999. Hyperglycemia causes oxi-
dative stress in pancreatic beta-cells of GK rats, a
model of type 2 diabetes. Diabetes. 48:927–932.
41. Silva, J.P., et al. 2000. Impaired insulin secretion
and β-cell loss in tissue specific knockout mice with
mitochondrial diabetes. Nat. Genet. 26:336–340.
42. Kaneto, H., et al. 2001. Activation of the hexos-
amine pathway leads to deterioration of pancreatic
beta-cell function through the induction of oxida-
tive stress. J. Biol. Chem. 276:31099–31104.
43. Sakuraba, H., et al. 2002. Reduced beta-cell mass
and expression of oxidative stress-related DNA
damage in the islet of Japanese Type II diabetic
patients. Diabetologia. 45:85–96.
44. Sakai, K., et al. 2003. Mitochondrial reactive oxygen
species reduce insulin secretion by pancreatic β-cells.
Biochem. Biophys. Res. Commun. 300:216–222.
45. Feltus, F.A., Lee, E.K., Costello, J.F., Plass, C., and
Vertino, P.M. 2003. Predicting aberrant CpG
island methylation. Proc. Natl. Acad. Sci. U. S. A.
46. Bachman, K.E., et al. 2003. Histone modifications
and silencing prior to DNA methylation of a tumor
suppressor gene. Cancer Cell. 3:89–95.
47. Kouzarides, T. 2002. Histone methylation in
transcriptional control. Curr. Opin. Genet. Dev.
48. Fuks, F., Burgers, W.A., Brehm, A., Hughes-Davies,
L., and Kouzarides, T. 2000. DNA methyltrans-
ferase Dnmt1 associates with histone deacetylase
activity. Nat. Genet. 24:88–91.
49. Ogata, E.S., Bussey, M., and Finley, S. 1986. Altered
gas exchange, limited glucose, branched chain
amino acids, and hypoinsulinism retard fetal
growth in the rat. Metabolism. 35:950–977.
50. Kaminsky, Z., Sun-Chong, W., and Petronis, A.
2006. Complex disease, gender, and epigenetics.
Ann. Med. 38:530–544.
51. Ke, X., et al. 2006. Uteroplacental insufficiency
affects epigenetic determinants of chromatin struc-
ture in brains of neonatal and juvenile IUGR rats.
Physiol. Genomics. 25:16–28.
52. Finegood, D.T., Scaglia, L., and Bonner-Weir, S.
1995. Dynamics of beta-cell mass in the growing
rat pancreas. Estimation with a simple mathemati-
cal model. Diabetes. 44:249–256.
53. Stoffers, D.A., Heller, R.S., Miller, C.P., and Habener,
J.F. 1999. Developmental expression of the home-
odomain protein IDX-1 in mice transgenic for an
IDX-1 promoter/lacZ transcriptional reporter.