Studies in the IUGR rat also demonstrate that fetal growth retardation induces epigenetic modifications of key genes regulating ß-cell development (125
-1 is a homeodomain-containing transcription factor that plays a critical role in the early development of both endocrine and exocrine pancreas, and then in the later differentiation and function of the ß cell. As early as 24 hours after the onset of growth retardation, Pdx-1
mRNA levels are reduced by more than 50% in IUGR fetal rats. Suppression of Pdx
-1 expression persists after birth and progressively declines in the IUGR animal, implicating an epigenetic mechanism.
Chromatin modification mechanisms serve a critical function in affecting the transcriptional status of genes. Our data demonstrate that the open chromatin domain marked by histone H3 and H4 acetylation at the proximal promoter of Pdx1 is essential for transcription. Robust Pdx1 expression in islets from control animals is coincident with the presence of acetylated histones H3 and H4 as well as trimethylated H3K4. Loss of these marks results in Pdx1 silencing and reversal of IUGR induced epigenetic modifications normalizes Pdx1 expression. These data suggest that histone modifications can be stably propagated throughout life.
The first epigenetic mark that is modified in ß-cells of IUGR animals is histone acetylation (). Islets isolated from IUGR fetuses show a significant decrease in H3 and H4 acetylation at the proximal promoter of Pdx1
. These changes in H3 and H4 acetylation are associated with a loss of binding of USF-1 to the proximal promoter of Pdx1
. USF-1 is a critical activator of Pdx1
transcription and decreased binding markedly decreases Pdx1
). After birth, histone deacetylation progresses and is followed by a marked decrease in H3K4 trimethylation and a significant increase in dimethylation of H3K9 in IUGR islets (). Progression of these histone modifications parallels the progressive decrease in Pdx1
expression as glucose homeostasis deteriorates and oxidative stress increases in IUGR animals. Nevertheless, in the IUGR pup (at 2 weeks of age) these silencing histone modifications alone suppress Pdx1
expression since there is no appreciable methylation in the CpG island and reversal of histone deacetylation in IUGR islets (in the presence of active ß-cell replication) is sufficient to nearly normalize Pdx1
Summary of epigenetic changes at Pdx1 in IUGR rats during the development of type 2 diabetes
The initial mechanism by which IUGR silences Pdx1 is by recruitment of co-repressors, including HDAC1 and mSin3A, which catalyze histone deacetylation - the first repressive mark observed at Pdx1 in IUGR islets. Binding of these deacetylases in turn facilitates loss of trimethylation of H3K4 further repressing Pdx1 expression (). Our observation that inhibition of HDAC activity by TSA treatment normalizes H3K4me3 levels at Pdx1 in IUGR islets suggests that the association of HDAC1 at Pdx1 in IUGR islets likely serves as a platform for the recruitment of a demethylase, which catalyzes demethylation of H3K4.
As described above, DNA methylation of a CpG island in the promoter is a key mechanism for silencing gene expression. Most CpG islands remain unmethylated in normal cells, however, under conditions of oxidative stress (116
), CpG islands can become methylated de novo
. This is particularly relevant to type 2 diabetes, as there are now substantial data that show that oxidative stress plays a significant role in the progression of ß-cell deterioration (131
). Further, IUGR induces mitochondrial dysfunction in the ß-cell leading to increased production of ROS and oxidative stress (104
). It is not known why particular CpG islands are susceptible to aberrant methylation. A study by Feltus et al (115
) suggests that there is a “sequence signature associated with aberrant methylation”. Of particular relevance to this study is their finding that Pdx1
and a flanking gene, Cdx
-2 (also encoding a homeobox protein), were two of only 15 genes (a total of 1749 genes with CpG islands were examined) that were methylation susceptible under conditions of increased methylation induced by overexpression of DNMT1.
The molecular mechanism responsible for DNA methylation in IUGR islets is likely to involve H3K9 methylation. A number of studies have shown that methylation of H3K9 precedes DNA methylation (136
). It has also been suggested that DNA methyltransferases may act only on chromatin that is methylated at lysine 9 on histone H3 (H3K9) (137
). Histone methyltransferases bind to the DNA methylases DNMT3A and DNMT3B thereby initiating DNA methylation (136
These results demonstrate that IUGR induces a self-propagating epigenetic cycle in which the mSin3A/HDAC complex is first recruited to the Pdx1 promoter, histone tails are subjected to deacetylation and Pdx1 transcription is repressed. At the neonatal stage, this epigenetic process is reversible and may define an important developmental window for therapeutic approaches. However, as dimethylated H3K9 accumulates, DNMT3A is recruited to the promoter and initiates de novo DNA methylation, which locks in the silenced state in the IUGR adult pancreas resulting in diabetes. Our studies indicate novel mechanisms of epigenetic regulation of gene expression in vivo which link gene silencing in the ß-cell to the development of type 2 diabetes and suggest novel therapeutic agents for the prevention of common diseases with late-onset phenotypes.
How do these events lead to diabetes? Targeted homozygous disruption of Pdx
-1 in mice results in pancreatic agenesis (reviewed in 138
), and homozygous mutations yield a similar phenotype in humans (138
). Milder reductions in Pdx
-1 protein levels, as occurs in the Pdx
+/− mice, allow for the development of a normal mass of ß cells (138
), but result in the impairment of several events in glucose-stimulated insulin secretion (138
). These results indicate that Pdx
-1 plays a critical role, distinct from its developmental role, in the normal function of ß cells (138
). This may be the reason that humans with heterozygous missense mutations in Pdx
-1 exhibit early and late onset forms of type 2 diabetes (138