Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1

Pdx1 is a pancreatic and duodenal homeobox 1 transcription factor that regulates pancreas development and β cell differentiation.

Both genetic and acquired reductions in Pdx1 expression in humans and in animal models have been shown to cause type 2 diabetes, 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 chromatin protein binding (15). The region spanning the proximal 5ι promoter 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 epigenetic 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 involving histone modifications, DNA methylation, chromatin remodeling, and transcription were responsible for Pdx1 silencing during the transition 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 recruitment of histone deacetylase 1 (HDAC1) and Sin3A to the proximal promoter of Pdx1. Loss of acetylation is accompanied by loss of binding of the key transcription factor, USF-1. As the disease state progresses, 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 knowledge 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 secondary 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 5ι 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 5ι region.

Methylation status of the Pdx1 gene promoter in IUGR and control islets.

The proximal 5ι flanking region of the Pdx1 gene and its first exon include a highly conserved CpG island that encompasses nucleotides –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 promoters are typically characterized by a repressed chromatin structure and hypermethylated CpGs. We determined the DNA methylation status of the portion of the CpG island that is located in the Pdx1 promoter in islets of IUGR and control animals using bisulfite treatment and pyrosequencing analyses. Evaluation of DNA from islets of 5 IUGR and 5 control animals revealed that at age 2 weeks none of the 14 CpG sites in the Pdx1 promoter were methylated in islets from either IUGR or control animals. At 6 months of age (n = 5 animals per group), Pdx1 DNA methylation levels across the CpG island averaged 51.3% ± 10.3% compared with no CpG methylation in controls (P < 0.05 vs. controls). No single CpG site was consistently methylated (Figure 1B).

Binding of DNA methyltransferases at Pdx1 in IUGR islets.

Mammalian CpG methylation is mediated by 3 active DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b. Dnmt1 mediates replication-coupled maintenance of DNA methylation patterns, whereas Dnmt3a and Dnmt3b are considered to be de novo DNA methylases (20, 21). Surprisingly, we observed some association of Dnmt1 to Pdx1 in islets of 2-week-old IUGR pups, despite the lack of DNA methylation of CpGs (Figure 2). However, there was no binding of Dnmt3a or Dnmt3b to Pdx1 at this age. At 6 months of age, when DNA methylation at Pdx1 is increased in IUGR islets, Dnmt1 binding was significantly enhanced (Figure 2) and moderate levels of Dnmt3a, but not Dnmt3b, were also associated with Pdx1 (Figure 2). No binding of Dnmt1 or Dnmt3a and -3b to Pdx1 was detected in control islets at either age.

Characterization of the histone code at the Pdx1 promoter.

Whereas DNA methylation is associated with transcriptional silencing, histone acetylation is associated with active chromatin, and deacetylation results in repressed chromatin structure (15). The impact of IUGR on the acetylation status of core histones H3 and H4, which are physically associated with the Pdx1 promoter, was determined by ChIP analyses using primers that encompass the region of the CpG island at Pdx1, shown in Figure 1A. Similar to findings in a β cell tumor line (17), this region of Pdx1 was heavily acetylated at H3 and H4 in islets from control animals (Figure 3). IUGR significantly reduced the abundance of acetylated H3 and H4 and deacetylation progressed with age. By 6 months of age, H3 and H4 acetylation was absent (Figure 3).

Trimethylation of lysine 4 at H3 (H3K4me3) preferentially marks promoters, is associated with active genes, and is influenced by density of H3 acetylation (22, 23). In islets from control animals at all ages, there was a high association of H3K4me3 at Pdx1. In contrast, despite the marked decrease in histone acetylation in IUGR fetuses, there was no difference in the methylation status of H3K4 between IUGR and control fetal animals. However, in IUGR animals at 2 weeks of age, the abundance of H3K4me3 was significantly reduced, and by 6 months of age, H3K4me3 was absent at the Pdx1 proximal promoter region (Figure 3).

Loss of H3K4 trimethylation can initiate dimethylation of lysine 9 at H3 (H3K9me2), a histone modification that is involved in the establishment and maintenance of silent heterochromatin regions (24). ChIP assays demonstrated that by 2 weeks of age, H3K9me2 at the Pdx1 promoter was present in IUGR islets and H3K9me2 binding increased with age (Figure 3). We did not observe methylation of H3K27, another silencing methyl mark (data not shown) in IUGR or control islets at any age.

Histone modifications inhibit USF-1 binding at Pdx1.

A number of studies have demonstrated that USF-1 binds to and functionally regulates the Pdx1 gene promoter (25, 26). A reduction in endogenous USF-1 binding leads to a significant decrease in Pdx1 promoter activity, which, in turn, results in marked reductions in Pdx1 mRNA and protein levels (25). The USF-1 binding site is highly conserved and is located at a CpG site within the CpG island (Figure 1A). As expected, we observed strong association of USF-1 at Pdx1 in control islets at all ages examined (Figure 4A). In contrast, USF-1 binding was abolished in IUGR islets at all ages. Western blot analyses showed no difference in USF-1 protein abundance between IUGR and controls indicating that decreased USF-1 association with Pdx1 was not due to a decrease in USF-1 protein levels in IUGR islets (Figure 4B).

Recruitment of transcription repressors to the Pdx1 promoter in IUGR islets.

The reduction of histone H3 and H4 acetylation in IUGR islets suggested that HDACs might be involved in facilitating the observed histone modifications. In fetal islets from IUGR animals, there was modest binding of HDAC1, but not mSin3A, at Pdx1. Neither protein was associated with the proximal promoter of Pdx1 in control fetal islets (Figure 5A). At 2 weeks and 6 months of age, we observed a robust association of HDAC1 and mSin3A at Pdx1, which was concomitant to complete deacetylation of H3 and H4 in IUGR islets (Figure 5, B and C). There was no binding of HDAC2 at Pdx1 in IUGR or control islets at any age (data not shown).

Inhibition of HDAC partially restores Pdx1 expression.

To determine whether either DNA methylation or histone deacetylation is sufficient for silencing of Pdx1 expression in IUGR animals, islets were isolated from 2-week-old IUGR and control animals. At this age (as opposed to later in life), there is significant replication of β cells in rodents making it the ideal age to determine whether stable propagation of histone modifications occurs during cell division and whether these changes can be reversed (27). Islets from IUGR and control animals were cultured for 5 days in the presence of 5-aza-2′-deoxycytidine (5-AzaC) and/or trichostatin A (TSA), which inhibit Dnmts and HDACs, respectively. TSA treatment alone (and in combination with 5-AzaC) normalized acetylation of H3 and restored methylation of H3K4 at the Pdx1 promoter in IUGR islets, further substantiating the dependence of H3K4 methylation upon levels of H3 acetylation (Figure 6, A and B). However, Pdx1 expression (Figure 6C) was largely but not completely restored in IUGR islets, indicating that other relatively minor mechanisms of repression might exist in addition to the recruitment of a histone deacetylation complex. While we did not observe any DNA methylation at the CpG island in IUGR islets, it is possible that other CpGs located in distal enhancer regions could contribute to Pdx1 repression. However, treatment with 5-AzaC alone did not alter Pdx1 mRNA levels in either IUGR or control islets (Figure 6C), suggesting that DNA methylation does not contribute to Pdx1 silencing at this young age.

Surprisingly, TSA, but not 5-AzaC, treatment resulted in normalization of USF-1 binding to Pdx1 in IUGR islets (Figure 6D). Thus, reversal of histone modifications by TSA normalized association of USF-1 to Pdx1 in the IUGR animal. Neither agent altered USF-1 protein levels (data not shown). USF-1 is a methylation-sensitive transcription factor and the USF site contains an evolutionary, conserved CpG. However, our data suggest that histone modifications, and not DNA methylation, are responsible for loss of USF-1 association with the proximal promoter of Pdx1 in IUGR islets.

Animal model.

Surgical methods have been previously described (4, 5, 49). In brief, time-dated Sprague-Dawley pregnant rats were individually housed under standard conditions and allowed free access to standard rat chow and water. On day 18 of gestation (term is 22 days), pregnant rats were anesthetized with intraperitoneal xylazine (8 mg/kg) and ketamine (40 mg/kg), and both uterine arteries were ligated. This results in an approximately 50% reduction in uteroplacental blood flow and induces IUGR (4, 49). Controls underwent sham surgery. Rats recovered within a few hours and had ad libitum access to food and water. The pregnant rats were allowed to deliver spontaneously, and the litter size was randomly reduced to 8 at birth to assure uniformity of litter size between IUGR and control litters. IUGR and control pups were fostered to unoperated normal female rats and remained with their foster mothers until they were weaned. Only male animals were studied to avoid the potentially confounding hormonal variables associated with female rats. While it is possible that the changes that we observed may differ in female rats as gender specific effects on histone acetylation and DNA methylation have been reported (reviewed in refs. 50, 51), we have not observed gender specific effects on β cell function in IUGR rats. Thus, it is unlikely that epigenetic modifications at Pdx1 may differ between male and female animals.

Islets were harvested from 10 litters each of 2-week-old IUGR and control animals, and islets were pooled from a litter. Islets were also harvested from 10 IUGR and 10 control animals at 6 months of age (each animal was from a different litter). In rats, β cells compose 95% of the cellular mass of the islet (52).

These studies were approved by the Animal Care Committee of the Children’s Hospital of Philadelphia and the University of Pennsylvania.

Pdx1 reporter gene analysis.

The generation and characterization of the Pdx1-LacZ transgenic reporter mouse was previously reported (53). A modification of the IUGR procedure was performed on gestation day 17 in maternal pregnant Pdx1-LacZ mice. Pancreatic islets were isolated from transgene-positive offspring at 3 weeks and RNA was isolated for real-time PCR quantification of Pdx1 and LacZ mRNA levels.

RNA isolation and real-time PCR.

Total RNA was extracted from islets using RNAzol B (Tel-Test Inc.). Real-time PCR was carried out using an ABI 7900HT Real-Time PCR system with SYBR Green Master Mix from Applied Biosystems. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase expression (primers are listed in Supplemental Table 1).

Methylation analysis of the Pdx1 CpG island.

Genomic DNA was extracted from islets of 2-week-old (n = 8 litters of 6 pups for each group), 6-month-old IUGR (n = 10), and control (n = 8) rats by phenol-chloroform-isoamyl alcohol and was collected by ethanol precipitation. We denatured 10 μg aliquots of XbaI-digested genomic DNA in 0.3 M NaOH at 37°C. Sulfonation and hydrolytic deamination reactions were then carried out by adding 2.5 M sodium bisulfite (Na₂S₂O₅)/10 mM hydroquinone solution (pH 5.0) to the digested genomic DNA and then incubating at 50°C for 4 hours in the dark. Bisulfite-converted DNA was purified using a QIAquick spin purification kit (QIAGEN) and eluted with tris/EDTA buffer (TE). Desulfonation reactions were performed in 0.3 M NaOH at 37°C for 15 minutes, and DNA was precipitated with 3 M sodium acetate/ethanol and resuspended in water.

The PCR reaction buffer for the pyrosequencing of Pdx1 contained 2.5 mM MgCl₂, 0.2 mM each of deoxyribonucleotide triphosphate (dNTP), 0.2 mM each of primer, and 2 U AmpliTaq Gold (Applied Biosystems) in a 25 ml reaction mixture. The PCR amplification was done for 50 cycles with an annealing temperature of 55°C (primers are listed in Supplemental Table 1). All the reactions were constructed as recommended by the manufacturer’s instructions (Biotage AB). The pyrosequencing reaction was performed automatically using a PSQ 96MA system along with a SNP Reagent kit. As a control, plasmid DNA, which is unmethylated, was amplified and then subjected to the same analysis in parallel in order to confirm the completion of the bisulfite reaction.

ChIP assays.

Islets were trypsin digested and suspensions were fixed in phosphate-buffered saline containing 1% formaldehyde and protease inhibitor cocktail. Sonication was performed to yield DNA fragments ranging in size from 200–1,000 bp, followed by centrifugation for 10 minutes. Soluble chromatin (150 μg) was diluted in IP buffer and protease inhibitors. Supernatants were collected and cleared by incubation with protein A–sepharose, sonicated salmon sperm DNA, and 10 μl of preimmune serum. An aliquot of supernatant was collected and used as the input DNA. IP was carried out overnight with IgG (negative control) or primary antibody to the modified histone (acetylated histone H3 and H4, trimethyl-histone H3 [Lys4], acetylated H3K4, and dimethyl-histone H3 [Lys9]), HDAC1 (Upstate Biotech), USF-1, Dnmt1, Dnmt3a, and Dnmt3b (Abcam), and mSin3A (Santa Cruz Biotechnology Inc.). Immune complexes were isolated by incubation with protein A–agarose, and the complexes were then serially washed in low-salt buffer, high-salt buffer, LiCl buffer, and finally TE. Bound complexes were eluted from protein A with a 1% SDS buffer and cross-links were reversed in the presence of Rnase A and extensively digested with proteinase K. The released DNA was extracted, precipitated, and resuspended in water. DNA sequences in the “input” and the IP samples were quantified relative to each other by real-time PCR (primers are listed in Supplemental Table 1).

To determine whether reversal of epigenetic modifications would restore Pdx1 expression in IUGR animals, islets were harvested from 2-week-old IUGR and control pups and cultured for 5 days in RPMI 1640 culture medium containing 11 mM glucose, supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 50 IU/ml penicillin, and 50 mg/ml streptomycin (Gibco BRL) in the presence and absence of 10 μM TSA and/or 1 μM 5-AzaC, which inhibited HDACs and Dnmt1, respectively.

Statistics.

Statistical analyses were performed using analysis of variance and the 2-tailed Student’s unpaired t test. A P value of less than 0.05 was considered significant.