Exendin-4 increases histone acetylase activity and reverses epigenetic modifications that silence Pdx1 in the intrauterine growth retarded rat
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The abnormal intrauterine milieu of intrauterine growth retardation (IUGR) permanently alters gene expression and function of pancreatic beta cells leading to the development of diabetes in adulthood. Expression of the pancreatic homeobox transcription factor Pdx1 is permanently reduced in IUGR islets suggesting an epigenetic mechanism. Exendin-4 (Ex-4), a long-acting glucagon-like peptide-1 (GLP-1) analogue, given in the newborn period increases Pdx1 expression and prevents the development of diabetes in the IUGR rat.
IUGR was induced by bilateral uterine artery ligation in fetal life. Ex-4 was given on postnatal days 1–6 of life. Islets were isolated at 1 week and at 3–12 months. Histone modifications, PCAF, USF1 and DNA methyltransferase (Dnmt) 1 binding were assessed by chromatin immunoprecipitation (ChIP) assays and DNA methylation was quantified by pyrosequencing.
Phosphorylation of USF1 was markedly increased in IUGR islets in Ex-4 treated animals. This resulted in increased USF1 and PCAF association at the proximal promoter of Pdx1, thereby increasing histone acetyl transferase (HAT) activity. Histone H3 acetylation and trimethylation of H3K4 were permanently increased, whereas Dnmt1 binding and subsequent DNA methylation were prevented at the proximal promoter of Pdx1 in IUGR islets. Normalisation of these epigenetic modifications reversed silencing of Pdx1 in islets of IUGR animals.
These studies demonstrate a novel mechanism whereby a short treatment course of Ex-4 in the newborn period permanently increases HAT activity by recruiting USF1 and PCAF to the proximal promoter of Pdx1 which restores chromatin structure at the Pdx1 promoter and prevents DNA methylation, thus preserving Pdx1 transcription.
KeywordsBeta cell Epigenetics Exendin-4 Histone Intrauterine growth retardation Pdx1
cAMP response element-binding protein
Histone acetyl transferase
Histone H3 lysine 4
Trimethylated histone H3 lysine 4
Histone H3 lysine 9
Intrauterine growth retardation
p300/CBP (CREB binding protein) associated factor
- PD 1–6
Postnatal day 1–6
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 . We have demonstrated that the abnormal intrauterine milieu associated with IUGR limits the supply of critical substrates and hormones and affects the development of the fetus by permanently modifying gene expression and function of susceptible cells, such as the pancreatic beta cell leading to the development of diabetes in adulthood [2, 3, 4].
Pdx1 encodes a homeobox transcription factor critically important for pancreatic beta cell function and development and plays a pivotal role in the development of diabetes in humans and rodents. Even a relatively modest decrease in Pdx1 expression impairs the compensatory response to insulin resistance [2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]. Expression of Pdx1 is permanently reduced in IUGR beta cells and in previous studies we demonstrated that aberrant histone modifications and later gain of DNA methylation at the proximal promoter of Pdx1 are responsible for decreased Pdx1 transcription .
The long-acting GLP-1 analogue, exendin-4 (Ex-4) increases Pdx1 expression in IUGR beta cells [4, 15, 16, 17, 18] and is being used to treat humans with type 2 diabetes. The molecular mechanisms by which Ex-4 increases Pdx1 transcription in IUGR animals are unknown. In previous studies, we found that administration of Ex-4 during the prediabetic neonatal period (postnatal days 1–6 [PD 1–6]) prevents the development of diabetes in IUGR animals by restoring expression of Pdx1 to normal levels .
One of the earliest molecular events involved in silencing the Pdx1 promoter in islets of IUGR animals is the loss of binding of the critical activator, USF1 . However, USF1 protein levels are not reduced in IUGR animals, and this leads us to posit that IUGR may induce post-translational modifications (e.g. phosphorylation), which prevent the association of USF1 with the Pdx1 promoter. Transcriptional activity of USF1 is dependent on phosphorylation at Ser257 [19, 20, 21, 22, 23]; and USF1 purified from rat liver, spleen and kidney is primarily present in the phosphorylated form in the nucleus .
In the present study, we hypothesised that normalisation of Pdx1 transcription by Ex-4 treatment is mediated by phosphorylation of USF1, which in turn mediates epigenetic changes involving histone modifications and DNA methylation at the proximal promoter of Pdx1.
For a complete description of the methods, please see the electronic supplementary material (ESM).
Four experimental groups from our previously reported rat model of IUGR were studied [2, 3]: (1) control pups treated with vehicle (PBS); (2) control pups treated with Ex-4 (Bachem, King of Prussia, PA, USA); 1 nmol/kg body weight injected subcutaneously for 6 days starting on day of life 1; (3) IUGR pups treated with vehicle; and (4) IUGR pups treated with Ex-4. On day of life 7, islets were harvested for neonatal experiments. For neonatal experiments, islets were harvested from 16 litters of IUGR and 16 litters of control animals and pooled from one litter for each experiment. Islets were used from 22 male adult animals from different litters for each treatment group. These studies were approved by the Animal Care Committee of The Children’s Hospital of Philadelphia.
Total RNA was isolated from islets (n = 5, all from different litters, per group) using RNAzol B (Tel-Test, Friendswood, TX, USA). Quantitative PCR was performed as previously described . Pdx1 expression was normalised to β-actin expression after assessment of a panel of housekeeping genes found this to be the most stable.
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed as previously described  using approximately 1,000 neonatal and 500 adult islets per experiment per group. Quantitative PCR was used to measure binding of USF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetylated H3 (Millipore, Billerica, MA, USA), H3K9me2 (Millipore), p300 (Millipore), DNA methyltransferase (Dnmt) 1 (Millipore) and H3K4me3 (AbCAM, Cambridge, MA, USA) at the promoters of Pdx1 and β-actin. Results were expressed as IP per total input for each experimental group and each experiment was normalised to the IP per total input value for the control vehicle experimental group for each antibody. Control vehicle groups were not compared across age groups. Standard methods of ChIP data normalisation were used to normalise individual sample immunoprecipitated DNA to both total input DNA for each experimental group and to a control sequence . For the adult experiments, ChIP was performed at 3–4 months for PCAF, 6 months for Dnmt1 and 9–12 months for AcH3, H3K4me3 and H3K9me2. For the histone modifications, data were independently analysed for the 9- and 12-month ChIP experiments (ESM Fig. 1, ESM Tables 1–3). The individual histone modification profiles were consistent at 9 and 12 months and thus these results were combined for the manuscript.
Islet HAT activity was measured from pooled islet samples from 1-week-old animals, approximately 1,000 islets per group, via colorimetric assay using cofactor acetyl CoA (Kamiya, Seattle, WA, USA). Measurements were made at baseline and at 1, 2 and 3 h. Total HAT activity was computed as area under the curve and normalised to control vehicle.
USF1 phosphorylation and PCAF coimmunoprecipitation
Islets were harvested from animals at 1 week of age and nuclear protein extracts were then prepared from islets. Nuclear extracts (10 μg of protein/reaction) were first immunoprecipitated with normal rabbit serum (negative control) or anti-USF1. Membranes were incubated with anti-phosphoserine (1:500 dilution) or PCAF (1:500 dilution) antibody, and immunoreactive proteins were visualised by incubation with horseradish peroxidase-linked donkey anti-rabbit secondary antibody (1:5,000 dilution) and the ECL Plus western blotting system (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions. Densitometric analyses were then performed and values were normalised to control samples treated with vehicle.
Methylation analysis of the Pdx1 CpG island
Genomic DNA was extracted from islets from 6–9 month old animals. Bisulfite modification was done using the Zymo Research EZ Methylation Gold kit (Irvine, CA, USA). Pyrosequencing analysis was performed by EpigenDx (Worchester, MA, USA).
Statistical analyses were performed using analysis of variance and Student’s unpaired t test not requiring equal variance between groups. A p value less than 0.05 was considered significant.
Ex-4 treatment restores Pdx1 mRNA levels in IUGR islets
Ex-4 treatment restores histone acetylation at the Pdx1 promoter
Effects of Ex-4 on trimethylation of H3K4
In previous studies, we observed increased abundance of H3K9me2 at the Pdx1 proximal promoter in islets from adult IUGR rats. Thus, to determine whether Ex4 prevented H3K9 dimethylation, ChIP assays were performed in both 1 week and adult islets. There was very little association of H3K9me2 at the Pdx1 promoter in all groups at 1 week of age (Fig. 4c). However, in adults, H3K9me2 was abundant at the Pdx1 promoter in IUGR islets and Ex-4 prevented this silencing histone modification (Fig. 4d; IV vs IEx: p = 0.044).
USF1 binding is restored in IUGR Ex-4 treated animals
USF1 binding and histone modifications at the β-actin promoter
Given the dramatic changes in USF1 binding, H3 acetylation and H3K4me3 binding in islet chromatin at the proximal Pdx1 promoter, we considered the possibility that these changes may have represented non-specific transcription factor/protein binding to chromatin induced by tissue preparation technique. To exclude this possibility, we measured binding of USF1 and enrichment of acetyl H3 and H3K4me3 at the β-actin promoter, a gene whose expression does not change in IUGR islets. In 1-week-old islets there was no change in USF1 binding at the β actin promoter in any experimental group (ESM Fig. 2; p > 0.10 for IV vs IEx, CV and CEx). In addition, we did not detect any differences in acetyl H3 or H3K4me3 enrichment at the β-actin promoter in adult animals in any of the four experimental groups (ESM Fig. 2: p > 0.10 for IV vs IEx, CV and CEx for both acetyl H3 and H3K4Me3 in adult islets). In order to ensure that the transcription factor/protein binding alterations that we observed at the Pdx1 promoter were not due to non-specific antibody binding, we measured IgG and no antibody binding in experiments using antibodies to acetyl H3, H3K4me3, USF1 and PCAF. There was a negligible amount of immunoprecipitate when the ChIP experiments were run without antibody or with IgG antibody, indicating that we were successfully measuring the proteins of interest compared with non-specific protein binding at the Pdx1 promoter (ESM Table 4).
Ex-4 treatment increases histone acetyl transferase activity in IUGR islets
PCAF association with USF1 at the proximal promoter of Pdx1
0.13 ± 0.004
1.29 ± 0.042
1.00 ± 0.068
1.13 ± 0.004
Methylation status of the Pdx1 gene promoter in IUGR and control islets
To determine the underlying mechanisms responsible for prevention of DNA methylation in Ex-4 IUGR islets, we examined the association of Dnmt1 to Pdx1. Mammalian CpG methylation is mediated by Dnmts. Dnmt1 mediates replication-coupled maintenance of DNA methylation patterns [36, 37] and previous studies showed marked association of Dnmt1 at Pdx1 in IUGR islets at 6 months of age . ChIP assays were performed on adult islets when DNA methylation at Pdx1 was first observed in IUGR islets. Dnmt1 binding was significantly enhanced in IUGR vehicle islets (17.1 ± 2.1-fold change over CV, p = 0.021; Fig. 8b), and we did not detect any significant association of Dnmt1 at Pdx1 in IUGR Ex-4, CV or CEx islets (Fig. 8b).
The major finding of our study was that a short treatment course of Ex-4 in the newborn period permanently reverses the aberrant epigenetic modifications at the proximal promoter of Pdx1 in IUGR islets, an animal model of type 2 diabetes. Normalisation of USF1 binding, H3 acetylation and H3K4 trimethylation at the Pdx1 proximal promoter allows for an open chromatin domain, creating an environment permissive of Pdx1 transcription that is necessary for proper beta cell function and development. These are the first studies to show that Ex-4, a drug now commonly used for treatment of type 2 diabetes, permanently reverses epigenetic modifications of a key beta cell gene, Pdx1, leading to increased transcription.
USF1 is a critical activator of Pdx1 transcription and decreased binding of USF1 markedly reduces or abolishes Pdx1 transcription [29, 30]. Other authors and we have shown that loss of USF1 binding leads to an increase in repressive chromatin modifications such as methylation of H3K9 and H3K27 [14, 38]. Furthermore, USF1 binding is important for generating and maintaining adjacent histone modifications associated with active chromatin structure . Here, we show that neonatal treatment with Ex-4 restores binding of USF1 thus allowing for an open chromatin domain and restoration of Pdx1 transcription in IUGR islets. In the beta cell, Ex-4 activates various signalling pathways in pancreatic beta cells, in particular cAMP, protein kinase A (PKA), Ca2+ and protein kinase B (PKB/Akt) leading to phosphorylation of a number of proteins . We found that IUGR markedly decreases serine phosphorylation of nuclear USF1, which is normalised by Ex-4 treatment. Increased phosphorylation in turn is associated with increased USF1 binding at Pdx1 in IUGR islets. As a number of studies have shown that serine phosphorylation is obligate for USF1 binding to DNA, it is likely that this is the mechanism underlying Ex-4 induced increased association at Pdx1 in IUGR islets [19, 20, 21, 22].
The first epigenetic mark that is modified in pancreatic beta cells of IUGR animals is histone acetylation . Islets isolated from IUGR fetuses show a significant decrease in H3 acetylation at the proximal promoter of Pdx1 . After birth, histone deacetylation progresses in IUGR animals. In this study, we found that Ex-4 administration during the newborn period normalises H3 acetylation to levels equal to or greater than those of control animals at 1 week of age. Ex-4 mediates this process by increasing HAT activity in IUGR islets to control levels. Our data show that PCAF is the primary histone acetyl transferase that catalyses acetylation at Pdx1. Ex-4 treatment of IUGR islets significantly increases PCAF binding at the proximal promoter of Pdx1. PCAF, also known as (p300/CBP [CREB-binding protein]-associated factor), was initially described as a member of an activating complex containing CREB and p300. However, in islets, PCAF binds to Pdx1 in the absence of p300. Other investigators have also shown that PCAF possesses HAT activity independent of CREB and p300 .
The mechanism by which Ex-4 increases PCAF binding at Pdx1 in IUGR islets is unclear. It is likely that during the period of increased beta cell replication that takes place in the neonatal period, Ex-4 increases association of PCAF and USF1 at the Pdx1 promoter and that these proteins together are responsible for increasing HAT activity and recruiting histone activating marks to the chromatin. This is in keeping with other studies that have recently shown that USF1 interacts directly with and recruits PCAF to the proximal promoter regions of a number of genes [22, 38, 41]. Our data indicate that in adulthood, the relative increase in PCAF binding in IUGR vehicle-treated islets is no longer present. However, the resulting increase in histone activating marks at the Pdx1 promoter is permanent thus leading to the permanent increase in Pdx1 expression. This implies that once histone acetylation is established, a mediator such as PCAF no longer needs to be present at the promoter.
After birth, in addition to persistent histone deacetylation, the IUGR state is further characterised by a marked decrease in H3K4me3 and an increase in H3K9me2, additional marks of repressed chromatin structure . In our studies of IUGR animals treated with Ex-4, by the adult age H3K4me3 at Pdx1 is restored to control levels and dimethylation of H3K9 is inhibited. Histone acetylation is the initial step in chromatin remodelling leading to increased gene transcription, which is followed by histone methylation (specifically H3K4 trimethylation) . We have previously shown that H3K4 methylation precludes methylation at lysine 9. Our in vivo findings support several in vitro studies showing that active chromatin states are maintained by H3K4 methylation, which opposes the lysine methylations that characterise inactive chromatin [42, 43].
The prevention of the aberrant chromatin structure observed in IUGR animals treated with Ex-4 is permanent. Increased acetylation of H3 and trimethylation of H3K4 in islets isolated from adult IUGR animals receiving a neonatal course of Ex-4 was maintained well into adulthood. In the normal rat, replication of existing beta cells and formation of new beta cells are higher during the newborn period than at any other time in postnatal life [44, 45, 46]. Thus, these histone modifications are somehow maintained during active beta cell replication. It is not known how histone modifications are propagated in daughter cells and it remains to be determined if histone modifications can serve as templates for reproducing these same structures on newly incorporated nucleosomes following cell replication.
Of major significance was our finding that neonatal Ex-4 treatment prevented DNA methylation at the CpG island within the proximal promoter of Pdx1, the final step in IUGR induced chromatin remodelling. In IUGR newborn islets, there is no DNA methylation at Pdx1; however, by 6 months of age, high levels of DNA methylation are found at the proximal promoter of Pdx1 in IUGR but the Pdx1 promoter remains unmethylated in control animals. DNA methylation is thought to be an essential step in ‘locking in’ the aberrant chromatin changes characteristic of IUGR animals.
The mechanisms by which Ex-4 prevents DNA methylation in IUGR islets are likely to be related to the prevention of H3K9 dimethylation, which in turn prevents recruitment of Dnmt1, the primary DNA methyltransferase that is associated with Pdx1 in IUGR islets . A number of studies have shown that methylation of H3K9 precedes DNA methylation [37, 47]. It has also been suggested that DNA methyltransferases may act only on chromatin that is methylated at lysine 9 on histone H3 (H3K9) . Histone methyltransferases bind to DNA methylases thereby initiating DNA methylation .
Of particular interest is the finding that neonatal Ex-4 treatment increases H3K4 trimethylation at the Pdx1 promoter in adult islets, but that this was not associated with an increase in Pdx1 expression. This apparent dissociation between the epigenetic mark and gene expression indicates that there are other yet unidentified mechanisms that are in place to prevent overexpression of any particular gene thus preventing changes in beta cell mass and preserving glucose homeostasis. Such braking mechanisms could include additional epigenetic regulators at the chromatin level or could function through the saturation of GLP-1 or other receptors. Further studies are needed to explore these possible regulatory mechanisms.
In conclusion, our results demonstrate that Ex-4 interrupts the self-propagating aberrant epigenetic cycle in IUGR islets by inducing PCAF and USF1 binding to the Pdx1 promoter, which in turn restores histone acetylation and H3K4 methylation, thereby preventing H3K9 and DNA methylation. At the neonatal stage, this epigenetic malprogramming is reversible and may define an important developmental window for innovative therapeutic approaches to prevent the development of adult onset diseases. Our studies indicate novel mechanisms by which Ex-4 functions to regulate gene expression in vivo. By increasing HAT activity, Ex-4 is able to reverse epigenetic modifications induced by IUGR and normalises Pdx1 expression.
This study was supported by the National Institutes of Health grant #DK55704 and #DK062965 (RAS) and the Lilly/Lawson Wilkins Pediatric Endocrine Research Fellowship and University of Pennsylvania Institute of Translational Medicine and Therapeutics Fellowship (SEP). We would like to thank H. Niu and F. Li for their technical expertise.
SEP designed the experiment, collected data, analysed data, wrote and revised the manuscript. LJJS collected data, analysed data and revised the manuscript. YH collected data, revised the manuscript and analysed data. DAS designed experiments, interpreted data and revised the manuscript. RAS designed experiments, analysed data, and wrote and revised the manuscript. All authors approved the manuscript in the final version.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript except for D.A.S. Stoffers who is a co-inventor on a patent entitled ‘Differentiation of non-insulin producing cells into insulin-producing cells by GLP-1 or exendin-4 or uses thereof’.
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