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Hepatic specification and functional maturation are tightly controlled throughout development. N6-methyladenosine (m6A) is the most abundant RNA modification of eukaryotic mRNAs and is involved in various physiological and pathological processes. However, the function of m6A in liver development remains elusive. Here we dissect the role of Mettl3-mediated m6A modification in postnatal liver development and homeostasis. Knocking out Mettl3 perinatally with Alb-Cre (Mettl3 cKO) induces apoptosis and steatosis of hepatocytes, results in severe liver injury, and finally leads to postnatal lethality within 7 weeks. m6A-RIP sequencing and RNA-sequencing reveal that mRNAs of a series of crucial liver-enriched transcription factors are modified by m6A, including Hnf4a, a master regulator for hepatic parenchymal formation. Deleting Mettl3 reduces m6A modification on Hnf4a, decreases its transcript stability in an Igf2bp1-dependent manner, and down-regulates Hnf4a expression, while overexpressing Hnf4a with AAV8 alleviates the liver injury and prolongs the lifespan of Mettl3 cKO mice. However, knocking out Mettl3 in adults using Alb-CreERT2 does not affect liver homeostasis. Our study identifies a dynamic role of Mettl3-mediated RNA m6A modification in liver development.
In this work, we generate hepatic-specific Mettl3 knockout (Mettl3 cKO) mice by crossing Mettl3flox/flox mice with Albumin (Alb)-enhancer/promoter driven-Cre transgenic mice to investigate the role of m6A modification in liver development. Hepatic perinatal loss of Mettl3 causes severe liver damage, including steatosis, apoptosis, and fibrosis, and finally results in lethality within 7 weeks. Using m6A-RNA immunoprecipitation (m6A-RIP) sequencing and RNA-sequencing, we identify that crucial liver-enriched transcription factors, including Hnf4a, are modified by m6A in liver development. Loss of Mettl3 induces depletion of m6A on Hnf4a transcripts, decreases its transcript stability in an Igf2bp1-dependent manner, and down-regulates Hnf4a expression, while overexpressing Hnf4a with AAV8 alleviates the liver injury and prolongs lifespan of Mettl3 cKO mice. However, deletion of Mettl3 in adult mouse livers using Albumin-enhancer/promoter-driven CreERT2 shows minimal effects on liver homeostasis. In conclusion, we elucidate a dynamic role of Mettl3-mediated RNA m6A modification during mouse postnatal liver development and decipher a novel function of epitranscriptomic control of liver organogenesis.
To study the role of m6A modification in liver development, we first tested the expression level of critical subunits of the m6A methyltransferase complex, Mettl3 and Mettl147. Both components showed shallow protein levels in mouse neonates (within one day after birth), increased gradually, and peaked at 2-3 weeks, and then decreased from 4 weeks onwards (Supplementary Fig. 1a). A similar trend was observed in human livers with high expression in children and a subsequent decline with age (Supplementary Fig. 1b). These results indicated that m6A is dynamically regulated in postnatal liver development. Global knockout of either Mettl3 or Mettl14 results in embryonic lethality caused by gastrulation defects19,20,21. Thus, to study the role of m6A modification, we generated mice with hepatic specific knockout of the catalytic subunit of the m6A methyltransferase complex, Mettl3, by crossing Mettl3flox/flox mice (with loxP sites flanking exons 2 and 4) with Alb-enhancer/promoter-driven Cre transgenic mice (Supplementary Fig. 1c, d). The specific knockout of Mettl3 in the liver was confirmed by genomic PCR, quantitative real-time PCR (RT-qPCR), western blot, and immunochemistry (Fig. 1a-d and Supplementary Fig. 1e-j). Genomic PCR and RT-qPCR showed that efficient knockout of Mettl3 started from day 1 after birth (Fig. 1b and Supplementary Fig. 1k), along with Cre expression (Supplementary Fig. 1i). As expected, livers from Mettl3 cKO mice showed a significant decrease in mRNA m6A levels compared to control mice (Supplementary Fig. 1l). In addition, we also observed that knocking out Mettl3 led to disruption of Mettl14 (Supplementary Fig. 1m), which is in accordance with previous reports19.
To gain further insights into the mechanism of Mettl3 regulating liver development, we conducted RNA-sequencing for liver tissues from Control and Mettl3 cKO mice at 1 day, 1 week, 2 weeks, and 4 weeks after birth. There were much more differentially regulated genes (DEGs) between Control and Mettl3 cKO mice at later time points (Supplementary Fig. 6a, Supplementary Dataset 3), which is consistent with our observations that Mettl3 cKO mice showed progressive severe liver damage 2 weeks after birth onward (Fig. 3a). Gene set enrichment analysis (GSEA) showed that targets of Hnf4a and Hnf1a were significantly repressed in Mettl3 cKO livers even at 1 day postnatally (Fig. 6a and Supplementary Fig. 6b). Dual-luciferase reporter assay and mutagenesis assay (Fig. 6b and Supplementary Fig. 6c, d) showed that co-transfection with WT, but not catalytic mutant Mettl322,23,24, significantly promoted luciferase activity in reporters carrying WT Hnf4a and Hnf1a fragments, while such increases were abolished when the m6A consensus motifs were mutated, confirming that the regulation of Hnf4a and Hnf1a by Mettl3 was indeed relying on m6A methylation of their transcripts. Both RT-qPCR and western blot confirmed that Hnf4a was downregulated in Mettl3 cKO livers at different time points postnatally (Fig. 6c-e). Although the RNA level of Hnf1a was downregulated at all time points (Supplementary Fig. 6e), we observed a dramatic decrease of Hnf1a protein with age and only observed a difference between Control and Mettl3 cKO mouse livers 1 week after birth (Supplementary Fig. 6f), indicating a less essential role of Hnf1a in the maturation of hepatocytes, which is consistent with previous studies25. Since Hnf4a is a master transcription factor required for liver development in both foetuses and adults and controls most aspects of mature hepatocyte function26,27, we mainly focused on Hnf4a for further studies. RNA-sequencing data showed that along with the downregulation of Hnf4a, most Hnf4a target genes, such as Apoa2, Apoc3, Cyp8b1, and Mttp, were repressed in Mettl3 cKO individuals (Supplementary Fig. 6g, Supplementary Dataset 3), which was validated by RT-qPCR (Supplementary Fig. 6h). We also noticed that Smad signaling, the central mediator of fibrosis28, was significantly enriched in Mettl3 cKO mouse liver tissues at 4 weeks (Supplementary Fig. 7a), supporting the phenomenon that massive liver fibrosis was induced in Mettl3 cKO animals (Fig. 4). These results indicate that Mettl3-mediated m6A controls the expression of crucial liver developmental genes during liver development.
m6A modification is involved in various aspects of RNA metabolism, including transcription, splicing, nuclear transportation, stability, and translation. Because we observed decreased expression of Hnf4a at both mRNA and protein levels, we determined the alternative splicing, nucleus-cytoplasm transportation, and mRNA stability of Hnf4a mRNA. Alternative splicing analysis showed no differences on Hnf4a transcripts in RNA-sequencing data from Control and Mettl3 cKO livers (Supplementary Dataset 4). The distribution of Hnf4a mRNA in nuclear and cytoplasm was also not affected by Mettl3 knockout (Supplementary Fig. 7b-d). Only mRNA stability showed significant changes in primary hepatocytes and the HepG2 cells with Mettl3 inhibition (Fig. 6f, g, and Supplementary Fig. 7e, f). Cells with Mettl3 deletion showed a shorter half-life of Hnf4a transcript, suggesting that Mettl3-mediated m6A controls the expression of Hnf4a at least partly by regulating its mRNA stability. To compare the global changes of mRNA stability after Mettl3 knockout, we subjected actinomycin D-treated hepatocytes from Control and Mettl3 cKO mice for RNA-sequencing (Supplementary Dataset 5). Consistent with previous reports15,19, knockout of Mettl3 enhanced mRNA stability globally, especially for m6A-modified genes (Supplementary Fig. 7g, h). Among genes involved in liver development (defined by Gene Ontology Resource, GO:0001889), only Hnf4a and another 10 genes showed decreased mRNA half-life when Mettl3 was knocked out, while most genes (including Cited2, Cebpa, Notch2, Dbp, et al.) were more stable or unchanged (Supplementary Fig. 7i and Supplementary Dataset 5). These results demonstrated that Mettl3 deficiency downregulated Hnf4a expression by reducing the half-life of Hnf4a mRNA.
To further strengthen our conclusion that Hnf4a is the primary mediator of Mettl3 function in liver development, we conducted rescue experiments using AAV serotype 8 (AAV8) to express Hnf4a under the control of a liver-specific promoter (thyroxine-binding globulin, TBG) (AAV8-TBG-Hnf4a) on Mettl3 cKO mice (Fig. 6j). Injection of AAV8-TBG-Hnf4a by superficial temporal vein on day two after birth successfully overexpressed Hnf4a in the liver (Supplementary Fig. 7m) and alleviated liver damage caused by hepatic Mettl3 knockout compared to AAV8-Ctrl at two weeks, evidenced by an increased number of Ki67+ proliferating hepatocytes and reduced hepatic steatosis (Fig. 6k-m). However, we did not see long-term benefits on mortality. This may attribute to the rapid dilution of AAV caused by the vigorous hepatocyte division within four weeks after birth29. Then we overexpressed Hnf4a by AAV-TBG-Hnf4a through tail vein injection at four-week-old Mettl3 cKO mice and found that Hnf4a overexpression significantly prolonged the life span of Mettl3 cKO mice (Fig. 6n). These results further demonstrated that Hnf4a is the primary factor mediating the function of Mettl3 in liver development. 1e1e36bf2d