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m6A modification-tuned sphingolipid metabolism regulates postnatal liver…
m6A modification-tuned sphingolipid metabolism regulates postnatal liver development in male mice
Introduction
Liver development
The liver is the primary site of haematopoiesis during embryogenesis but converts to a major metabolic organ in the adult, concomitant with extensive epigenetic modification
Hepatocytes, which are highly proliferative in the fetus, become quiescent, undergo hypertrophic growth
Diverse transcriptional and epigenetic mechanisms ensure that lineage specification, tissue growth and functional maturation occur precisely
Status
much less is known about the regulation of liver development and functional maturation during postnatal stages.
RNA modifications
m6A deposition is catalysed by the RNA methyltransferase complex containing methyltransferase-like 3 (Mettl3), Mettl14 and Wilms’ tumour 1-associating protein
conditional knockout (KO) of Mettl3 leads to cerebellar hypoplasia and axia-like movement disorders
Mettl3 also plays an essential role in the postnatal development of interscapular brown adipose tissue and adaptive thermogenesis
In addition, Mettl3-deficient naïve T cells fail to undergo homeostatic expansion and differentiation whereas Mettl3 deficiency impairs T follicular helper (TFH) differentiation and germinal centre responses
Status
the exact role of Mettl3/m6A-mediated epitranscriptomic control in postnatal liver development remains controversial.
why does prenatal-onset other than adult-onset Mettl3 deletion in the liver cause liver damage?
When is the critical period of time for this effect?
How does Mettl3 maintain physiological homeostasis during postnatal liver development?
Results
Q1
Mettl3 is enriched in embryonic and neonatal liver and declines during postnatal liver development
first analysed a publicly available transcriptomic dataset of livers from C57BL/6J mice across 12 time points
PCA
k-Means clustering analysis & time-course analysis was conducted to investigate their expression dynamics
Most known fetal liver-specific genes, including Afp and Igf2 accumulated in subclusters 4 and 5, were highly expressed in embryonic and neonatal livers and downregulated with age
measured the expression levels of Mettl3 and Mettl14, as well as other factors involved in m6A biology, in mouse livers at different ages
Mettl3, Mettl14 and Fto gradually downregulated after birth, accompanying the upregulation of genes implicated in hepatic metabolism, while Afp was shut down at week 2 after birth
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Hepatic Mettl3 deficiency induces hepatocyte hypertrophy, liver injury and growth retardation
generated liver-specific Mettl3 KO mice
RT–qPCR and immunoblotting analysis & RNA dot-blot analysis detect efficient Mettl3 depletion
Microscopic examination
Both terminal uridine nucleotide end-labelling (TUNEL) and cleaved Caspase-3 (cl-Casp3) IHC
Analysis of pro-survival signalling
adeno-associated virus serotype 8 (AAV8) carrying a thyroid-binding globulin (TBG) promoter-driven Cre recombinase into 8-week-old Mettl3-floxed mice
Immunoblotting and IHC analysis confirmed efficient Mettl3 KO
AAV8-Cre injection was carried out at 1 week after birth
Q2
Hepatic Mettl3 deficiency leads to metabolic reprogramming characterized by deregulated sphingolipid biosynthesis
compared the transcriptomes of Mettl3ΔHep versus wild-type (WT) livers
Differential expression analysis (false discovery rate (FDR) < 0.01, fold change (FC) > 1.5) identified 833 upregulated genes and 654 downregulated genes
Gene ontology (GO) analysis
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
RT–qPCR
immunoblotting and IHC
Mettl3 regulates Smpd3 expression by m6A-mediated RNA decay
methylated RNA immunoprecipitation combined with high-throughput sequencing (m6A–seq) on Mettl3ΔHep and WT livers
plotted m6A peak data against RNA sequencing (RNA-seq) data
Pathway analysis
the GGAC motif in eight genes in the 16-gene panel associated with sphingolipid metabolism
Mettl3 deficiency in hepatocytes results in ceramide accumulation, mitochondrial damage and endoplasmic reticulum stress
we measured the liver contents of several ceramide and SM species by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) in Mettl3ΔHep versus WT livers
higher plasma membrane fluidity in primary hepatocytes from Mettl3ΔHep versus WT mice
Electron microscopy
Pharmacological Smpd3 inhibition, Smpd3 knockdown or Sgms1 overexpression that counteracts Smpd3 ameliorated mitochondrial damage, endoplasmic reticulum stress and liver injury in Mettl3ΔHep mice
Hypothesis
Q1: Whether Mettl3 involves in postnatal liver development
Q2: How is this?
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