is a critical regulator of β cell growth and function in both fetal and postnatal developmental stages, and even a relatively modest decrease in expression impairs the compensatory response to insulin resistance (8
). Decreased Pdx1
expression plays a pivotal role in the development of diabetes in IUGR animals, as normalization of Pdx1
expression is associated with long-term maintenance of β cell mass and normal glucose homeostasis (6
). Uteroplacental insufficiency, the most common cause of IUGR, limits the supply of critical substrates such as oxygen, glucose, and amino acids to the fetus, resulting in an altered redox state and oxidative stress (4
). Here we have shown that this altered metabolic milieu decreases Pdx1
transcription by mediating a cascade of epigenetic modifications culminating in silencing of Pdx1
. These current studies are the first to our knowledge to characterize the histone code at Pdx1
in vivo in primary islets and link the progression of epigenetic modifications at a key gene to the development of diabetes.
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 H3K4me3. 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 β cells of IUGR animals is histone acetylation (Figure ). 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 H3K9me2 in IUGR islets (Figure ). 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 β 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 incomplete restoration of Pdx1
mRNA levels associated with the complete reversal of histone deacetylation in newborn IUGR islets suggests that there may be an additional repressor protein(s) such as SIRT4 or a microRNA like miR9 (both are expressed in islets; refs. 29
) that may be involved in chromatin silencing. Identifying such factors will require extensive future experiments. Alternatively, remodeling of active chromatin may only occur in β cells capable of replication, and there may be a minority of cells for which this does not occur, accounting for the extensive but incomplete reactivation of Pdx1
The initial mechanism by which IUGR silences Pdx1
is by recruitment of corepressors, including HDAC1 and mSin3A, which catalyze histone deacetylation — the first repressive mark observed at Pdx1
in IUGR islets. Binding of these deacetylases in turn facilitated loss of H3K4me3, further repressing Pdx1
expression (Figure ). Our observation that inhibition of HDAC activity by TSA treatment normalized 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. Lysine demethylases in the Jumonji class remove H3Kme3 and H3Kme2 (reviewed in ref. 31
), while LSD1 removes H3Kme1/2 (32
). Klose and coworkers have recently demonstrated that the retinoblastoma binding protein, RBP2, contains a JmjC domain, which can specifically demethylate H3K4me3 (33
). However, enzymes in the Jumonji class may not catalyze H3K4me3 demethylation in IUGR islets, as activity of these proteins is dependent upon the presence of an iron-binding domain (34
), which would be inactivated under conditions of oxidative stress that are present in IUGR islets (5
). These results imply that another class of histone demethylases may exist in β cells.
Loss of H3K4me3 was concomitant with a marked increase in H3K9me2 at Pdx1
in 2-week-old IUGR animals, suggesting that K4 methylation precludes methylation at lysine 9. These in vivo findings support several in vitro
studies showing that active chromatin states are maintained by H3K4 methylation, which opposes the lysine methylations that characterize inactive chromatin (36
). Since restoration of histone acetylation by TSA treatment of IUGR islets reversed H3K9me2, this also demonstrates that histone acetylation prevents methylation of H3K9. Thus, IUGR-induced histone modifications are mutually reinforcing and interdependent.
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 some normal circumstances, such as for imprinted genes, genes on the inactive X chromosome in females, and for some disease processes such as cancer (38
) and oxidative stress (39
), 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 β cell deterioration (40
). Further, IUGR induces mitochondrial dysfunction in the β cell leading to increased production of ROS and oxidative stress (5
). It is not known why particular CpG islands are susceptible to aberrant methylation. A recent study by Feltus et al. (45
) 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, Cdx2
(also encoding a homeobox protein), were 2 of only 15 genes (a total of 1,749 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 (21
). It has also been suggested that Dnmts may act only on chromatin that is methylated at lysine 9 on histone H3 (H3K9) (47
). Histone methyltransferases bind to the DNA methylases DNMT3A and DNMT3B, thereby initiating DNA methylation (21
). Surprisingly, we found that DNMT1 was associated with Pdx1
prior to CpG methylation. Since DNMT1 can be recruited by interaction with a HDAC such as HDAC1 (48
), which is already associated with the Pdx1
promoter in fetal IUGR islets, we suggest that this occurs in the IUGR islet prior to the alterations in histone methylation that only occur after birth (Figure ). As H3K9 methylation occurs during postnatal life in IUGR islets, this would then allow recruitment of the de novo methyltransferase DNMT3A (21
) (but not DNMT3B). Subsequent to onset of DNA methylation, DNMT1 would then be positioned to maintain the methylated state, locking in Pdx1
silencing in adult IUGR islets (Figure ).
In conclusion, our 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 H3K9me2 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. We believe our studies indicate novel mechanisms of epigenetic regulation of gene expression in vivo, which link gene silencing in the β cell to the development of type 2 diabetes and suggest therapeutic agents that we believe are novel for the prevention of common diseases with late-onset phenotypes.