The miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain controls neonatal metabolic adaptation

Abstract

In mammals, birth entails complex metabolic adjustments essential for neonatal survival. Using a mouse knockout model, we identify crucial biological roles for the miR-379/miR-410 cluster within the imprinted Dlk1-Dio3 region during this metabolic transition. The miR-379/miR-410 locus, also named C14MC in humans, is the largest known placental mammal-specific miRNA cluster, whose 39 miRNA genes are expressed only from the maternal allele. We found that heterozygote pups with a maternal--but not paternal--deletion of the miRNA cluster display partially penetrant neonatal lethality with defects in the maintenance of energy homeostasis. This maladaptive metabolic response is caused, at least in part, by profound changes in the activation of the neonatal hepatic gene expression program, pointing to as yet unidentified regulatory pathways that govern this crucial metabolic transition in the newborn's liver. Not only does our study highlight the physiological importance of miRNA genes that recently evolved in placental mammal lineages but it also unveils additional layers of RNA-mediated gene regulation at the Dlk1-Dio3 domain that impose parent-of-origin effects on metabolic control at birth and have likely contributed to mammal evolution.

Keywords: epigenetic; genomic imprinting; metabolic adaptation; microRNA; mouse model.

Figure 1. Targeted deletion of the miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain

1. Schematic representation of the ˜1-Mbp imprinted Dlk1-Dio3 region on mouse distal chromosome 12. Paternally expressed protein-coding genes (Dlk1, Rtl1, and Dio3) are symbolized by blue rectangles, while maternally expressed miRNA and C/D snoRNA genes are depicted by pink stem loops and ovals, respectively. Gtl2 (pink rectangle) is a long, maternally expressed non-coding RNA (ncRNA) gene. Anti-Rtl1, Rian, and Mirg correspond to poorly characterized maternally expressed ncRNAs from which some, but not all, miRNAs and C/D snoRNAs are processed. It should be noted, however, that some deposited RNA sequences may simply represent RNA species whose functionality, if any, remains questionable (Chiang et al, 2010). Arrows indicate the sense of transcription, with the horizontal broken line highlighting the notion that Gtl2, anti-Rtl1, Rian, and Mirg may belong to the same transcription unit. Differentially methylated regions, including the imprinting center (Ig-DMR) that controls imprinted expression over the domain, are indicated by filled and open lollipops (methylated and un-methylated, respectively). The relative positions of hairpin-like (pre-miRNA) structures within the miR-379/miR-410 cluster are indicated in the enlarged inset. Note that most pre-miRNA genes at the 3′ end of the cluster are positioned within introns of Mirg (gray rectangles and dotted lines represent exons and splicing events, respectively).

2. Top: The tissue-specific expression pattern of miRNAs was assayed by Northern blot analysis of adult mouse tissues as indicated on the panel, using a mixture of ³²P-labelled oligonucleotides antisense to some miRNAs scattered along the cluster (miR-411, 323, 376b, 376a, 134, 154, and 410). The same membrane was probed with a let-7 oligo probe (gel loading control). Bottom: The tissue-specific expression pattern of Mirg host gene transcripts was assayed by RT–qPCR relative to Gapdh using the same set of tissues. Note that Mirg expression, but not that of miRNAs, is detected in testes, indicating that post-transcriptional regulation may occur in this tissue. WAT: white adipose tissue. Data are expressed as mean ± s.e.m.

3. Cre/loxP-mediated site-specific deletion of the miR-379/miR-410 cluster. Left: Targeting strategy for disrupting the miR-379/miR-410 cluster through two independent homologous recombination events. Genome coordinates: UCSC Genome Browser, mouse, NCB137/mm9. Red and brown arrows indicate loxP and FRT recognition sites for the Cre and Flp site-specific recombinases, respectively. Right: PCR confirmation of the deletion using appropriate P1, P2, and P3 primers. The sequence of the deleted region was further confirmed by DNA sequencing. ΔMat and ΔPat represent heterozygous individuals with maternally and paternally inherited deletions, respectively, while WT correspond to wild-type littermate controls. M: DNA ladder (bp).

Source data are available online for this figure.

Figure 2. Targeted deletion of the miR-379/miR-410 cluster does not affect the expression levels of flanking genes

1. Expression of the miR-379/miR-410 cluster and its flanking C/D snoRNA (MBII-78, MBII-48) and miRNA (miR-127) genes was assayed by Northern blot analysis of the indicated samples (two individuals per genotype).

2. Expression of the miR-379/miR-410 cluster was assayed by Northern blot analysis of the dissected tissues indicated above the panels from WT or ΔMat E18.5 embryos. A tRNA-specific probe was used as the internal loading control.

3. Expression of selected miRNAs (miR-379, miR-376a, and miR-410) was assayed by RT–qPCR relative to U6 snRNA in P0 tissues prepared from WT or ΔMat individuals (n = 3 per genotype), as indicated below the histograms. BAT: brown adipose tissue. Data are expressed as mean ± s.e.m.

4. Expression of mRNAs (Dlk1 and Dio3) and mRNA-like transcripts (Gtl2, Rian, Mirg) was assayed by RT–qPCR relative to Gapdh mRNA in the indicated tissues. Blue and pink bars represent expression levels in ΔPat and ΔMat individuals, respectively (six individuals per genotype). Expression levels of WT were arbitrarily set to 1.

Source data are available online for this figure.

Figure 3. Maternal, but not paternal, deletion of the miR-379/miR-410 cluster impairs hepatic glycogenolysis and gluconeogenesis

A FSerum glucose (A, B), hepatic glycogen (C, D), insulin (E), glucagon (F) levels in WT (black), ΔMat (pink), severely hypoglycemic ΔMat (dark pink) or ΔPat (blue) neonates were measured in vaginally delivered neonates (P0, P1) or after cesarean delivery (E19.5, E19.5 + 1, E19.5 + 4). Insulin and glucagon levels of ΔMat individuals in the same cohort with the highest and lowest glucose concentration, denoted as high (50.78 ± 1.42 mg/dl) and low (27.38 ± 3.72 mg/dl), respectively, are also shown in the histograms to the right (E and F). Numbers of individuals analyzed are indicated above the histograms. G Glucose injections rescue the neonatal lethality phenotype. Dotted lines represent the % of ΔMat neonatal survival as observed from dead pups collected perinatally (Table 3). H Left: The lifespan of ΔMat neonates (pink line) is reduced relative to WT (black line) when P0 neonates were kept separated from their mother. Right: Glucose injections extend the lifespan of ΔMat neonates. Histograms show the survival rate of NaCl- or glucose-injected pups at 10 and 17 h post-injection. I The temporal expression of the gluconeogenic Pck1 and G6pc genes in liver (relative to Gapdh) was assayed by RT–qPCR (6 individuals per genotype). Data information: Data are expressed as mean ± s.e.m.

Figure 4. Maternal, but not paternal, deletion of the miR-379/miR-410 cluster impairs hepatic ketogenesis

A–H Serum β-hydroxybutyrate (A), serum fatty acids (B), serum triglycerides (C), hepatic neutral lipids (D), hepatic triglycerides (E), hepatic cholesterol (F), hepatic esterified cholesterol (G), and stomach milk (H) levels were measured in WT (black), ΔMat (pink), severely hypoglycemic ΔMat (dark pink), or ΔPat (blue) individuals at E19.5 after cesarean delivery or P0 and P1 after vaginal delivery. I Temporal expression of the ketogenic Cpt1a and Hmgcs2 genes was assayed in the liver (relative to Gapdh) by RT–qPCR (6 individuals per genotype). J The complete hepatic beta-oxidation of ¹⁴C-labelled palmitate at P0 was determined by the release of ¹⁴C-labelled CO_2. Beta-oxidation activities for WT were set to 1. Data information: Data are expressed as mean ± s.e.m.

Figure 5. Loss of the miR-379/miR-410 cluster is associated with major changes in the neonatal hepatic gene expression program

1. Top: Venn diagrams showing the number of up- and down-regulated genes revealed by Agilent microarrays at E19.5, E19.5 + 4, and P0 in ΔMat and WT livers (BH-adjusted P-value < 0.05). Bottom: Top 30 of up- and down-regulated genes. ncRNA genes and other poorly characterized transcripts, except the microRNA host gene (Mirg), were omitted from this microarray list.

2. Expression levels of selected misregulated genes validated by RT–qPCR. Blue and pink bars represent expression levels in ΔPat and ΔMat individuals, respectively. WT expression levels were arbitrarily set to 1.

3. Heatmap and hierarchical clustering of genes regulated in ΔMat vs. WT newborn livers at E19.5 or P0 (2,147 unique probes with BH-adjusted P-value < 0.05). Red: up-regulated; green: down-regulated; black: no change.

4. Perinatal expression of some genes was assayed by RT–qPCR analysis. Black and pink bars indicate expression levels in ΔPat and ΔMat individuals, respectively. Dark pink represents the most hypoglycemic mutant neonates at P1. WT expression levels at P0 were arbitrarily set to 1. Note that the dramatic decrease in Fgf21 mRNA levels in P1 hypoglycemic neonates may reflect the fact that fasting neonates display lower levels of Fgf21 expression (Hondares et al, 2010).

Data information: Data in (B–D) are expressed as mean ± s.e.m. (n = 4–6 individuals per genotype).

Figure 6. The miR-379/miR-410 cluster controls metabolic adaptation at the transition from fetal to postnatal life

Our model implies that maternally inherited deletion of the miR-379/miR-410 cluster interferes with the activation (or maintenance) of the neonatal hepatic gene program at the transition from fetal to postnatal life. This results in interlaced defects in the newborn (P0–P1) liver that include inefficient mobilization of hepatic glycogen stores, defective gluconeogenesis pathways and the inability to compensate with elevated levels of ketone bodies. Such a pleiotropic maladaptive metabolic response—in the absence of any apparent defects in pancreatic hormonal release—leads to severe neonatal hypoglycemia which most likely represents the main, if not the sole, cause of subsequent death.