m6A modification-tuned sphingolipid metabolism regulates postnatal liver development in male mice

Introduction

Liver development

RNA modifications

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.

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

Hypothesis

Q1: Whether Mettl3 involves in postnatal liver development

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

immunohistochemistry (IHC) analysis

RNA dot-blot analysis

Q2: How is this?

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Q2

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

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

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Mettl3 deficiency in hepatocytes results in ceramide accumulation, mitochondrial damage and endoplasmic reticulum stress

Pharmacological Smpd3 inhibition, Smpd3 knockdown or Sgms1 overexpression that counteracts Smpd3 ameliorated mitochondrial damage, endoplasmic reticulum stress and liver injury in Mettl3ΔHep mice

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

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