Allowed and disallowed genes. (A) Microarray expression profiles of pyruvate carboxylase (Pcx) versus lactate dehydrogenase A (Ldha), which are, respectively, highly expressed and deeply repressed in adult mouse pancreatic beta-cells. Mean mRNA expression signals ±SEM of three to five biological replicate microarray experiments. (B) Glucose levels in the blood are measured by the beta-cell through conversion of glucose to pyruvate, which is further metabolized in the mitochondria for ATP generation and anaplerosis. Downstream signals lead to insulin secretion. In many other cells, pyruvate does not necessarily come from glucose uptake. Extracellular pyruvate can be directly taken up by monocarboxylate transporter 1 (Mct1), or lactate can be converted to pyruvate by Ldha. However, when this happens in beta-cells, the glucose-sensing mechanism is disrupted, leading to inappropriate insulin release (Otonkoski et al. 2007). (C) Microarray expression profiles of 3-hydroxy 3-methylglutaryl-CoA synthase 2 (Hmgcs2) versus 2-oxoacid CoA transferase (Oxct1), which are, respectively, highly expressed and deeply repressed in adult liver. Mean mRNA expression signals ±SEM of three to five biological replicate microarray experiments. (D) In the fasted state, ketone bodies are generated in the liver. The first and rate-limiting enzyme for ketogenesis is Hmgcs2. Ketone bodies are transported in the blood and can be used by many organs as an alternative energy source. Oxct1 plays a central role in extrahepatic ketone body catabolism by catalyzing the reversible transfer of coenzyme A from succinyl-CoA to acetoacetate.

Epigenetic mechanism for islet- and liver-specific silencing of repressed genes. Histone H3 lysine9-acetylation (green bars) and lysine27-trimethylation (red bars) of chromatin around promoter region of repressed genes were compared in adult mouse tissues with and without observed repression. (A,B) For Ldha and Mct1, a high level of H3 lysine27-trimethylation is seen in islets but not in liver, whereas H3 lysine9-acetylation is more extensive in the liver. This difference parallels differences between liver and islets for mRNA expression (see also Fig. 2). (C) Histone modifications during postnatal liver maturation (age 1, 14, and 42 d and adult). Histone H3 trimethylation in the Oxct1 promoter increases during liver development and is significantly higher in adult liver than in adult brain, while acetylation at the lysine9 residue is lower for all liver time points versus brain. (D) Nucleosomes of the beta actin (Actb) promoter, which is active in all tissues, display a high level of histone H3 lysine9 acetylation and a low level of histone 3 lysine 27 trimethylation in islets, brain, and liver. (Black bars) IgG immunoprecipitation control. Data are means ± SEM of three to four QPCRs.

Tissue-specific repression of housekeeping genes. (A) Frequency distribution of the sum score for islets (green) and liver (brown) as baseline. Sum score numbers are calculated genewise and are the addition of the number of pairwise t-tests where expression in baseline is significantly lower (−1) or higher (+1) as compared to the other tissues. Liver- and islet-specific genes reside in the category with the highest possible sum score (right), whereas genes of which expression is specifically repressed in islets and liver are found in the category with the lowest possible sum score (left). (B) Strategy to identify genes that are selectively repressed in one tissue, based on mRNA profiling of 21 mouse tissues. Expression of a gene in a baseline tissue was compared to expression of that gene in all other tissues. When significantly lower compared to all other tissues, this gene was termed repressed in that tissue. This procedure was applied to all genes and repeated for all tissues in the panel. Statistical testing methods are described in Methods. (C) Pie-chart distribution of the number of specifically repressed genes in tissues. (D) Significant associations of specifically repressed genes (in all tissues) with molecular and cellular functions. The Benjamini-Hochberg-corrected P-value threshold of 0.05 is indicated by a dashed line.

Tissue-specific gene expression and repression of certain housekeeping genes during maturation of rat islets (A,B) and mouse liver (C,D). (A) mRNA expression level in extracts from rat pancreatic islets from age 1 d (P1) to 4 wk (P28) after birth measured by real-time qPCR; data were normalized for signal in islets of adult animals (10 wk; dashed line). Maturation-dependent up-regulation of genes that are important for beta-cells, such as insulin 2 (Ins2) and pyruvate carboxylase (Pc), was observed during this period. (B) In parallel we observed maturation-dependent repression of monocarboxylate transporter 1 (Mct1) and lactate dehydrogenase (Ldha), genes that are profoundly repressed in adult mouse islets (Table 1). (C,D) mRNA expression level in extracts from mouse fetal and neonatal liver between embryonic day E15.5 and 6 wk after birth. A progressive up-regulation (linear increasing trend P < 0.001) of Hmgcs2 (C) mirrors a progressive loss (linear decreasing trend P < 0.001) of expression of Oxct1 (D). Data in panels A–D are means ± SEM of three to four individual experiments. Statistical significance of the difference between postnatal and adult expression signal was calculated by one-way ANOVA with a Dunnett post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

MicroRNA 122 (miR-122) contributes to liver-specific silencing of Oxct1 expression. (A) Time course of miR-122 expression in vivo during liver maturation as measured by qPCR. miR-122 increases with a linear trend, P = 0.01. Statistical significance of the difference between P42 and other time points was calculated by one-way ANOVA with a Dunnett post-hoc test. (B) Treatment of in vitro differentiated BMEL cells with antagomiR-122 (α-miR-122) or a mutated antagomiR (α-miR-122 mut) control. Changes in miR-122 expression (left) and Oxct1 expression (right) were measured by Q-PCR. MiR-122 expression decreases sixfold by treatment with antagomiR-122 (α-miR-122). A mutated antagomiR (α-miR-122 mut) control did not significantly influence miR-122 expression. Oxct1 expression is increased by miR-122 knockdown with antagomiR-122, indicating that miR-122 inhibits Oxct1 expression. A mutated antagomiR did not significantly alter Oxct1 expression. Data are means ± SEM of three individual experiments. (C) Oxct1 RNA and protein expression in livers at E15.5 of wild-type versus liver-specific miR-122 overexpressing transgenic mice. A 1.7 ± 0.07-fold overexpression of miR-122 (mean ± SEM; n = 4 [wild-type] and n = 6 [transgenic]) was associated with a significant (t-test, P < 0.05) repression of Oxct1 RNA and protein, as determined by Q-RT-PCR (n = 4 or 6 for wild-type or transgenic, respectively) and Western blotting (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.