Analysis of diet-induced differential methylation, expression, and interactions of lncRNA and protein-coding genes in mouse liver

Jose P Silva,Derek Van Booven

doi:10.1038/s41598-018-29993-4

Abstract

Long non-coding RNAs (lncRNAs) regulate expression of protein-coding genes in cis through chromatin modifications including DNA methylation. Here we interrogated whether lncRNA genes may regulate transcription and methylation of their flanking or overlapping protein-coding genes in livers of mice exposed to a 12-week cholesterol-rich Western-style high fat diet (HFD) relative to a standard diet (STD). Deconvolution analysis of cell type-specific marker gene expression suggested similar hepatic cell type composition in HFD and STD livers. RNA-seq and validation by nCounter technology revealed differential expression of 14 lncRNA genes and 395 protein-coding genes enriched for functions in steroid/cholesterol synthesis, fatty acid metabolism, lipid localization, and circadian rhythm. While lncRNA and protein-coding genes were co-expressed in 53 lncRNA/protein-coding gene pairs, both were differentially expressed only in 4 lncRNA/protein-coding gene pairs, none of which included protein-coding genes in overrepresented pathways. Furthermore, 5-methylcytosine DNA immunoprecipitation sequencing and targeted bisulfite sequencing revealed no differential DNA methylation of genes in overrepresented pathways. These results suggest lncRNA/protein-coding gene interactions in cis play a minor role mediating hepatic expression of lipid metabolism/localization and circadian clock genes in response to chronic HFD feeding.

Highlights

More than 70% of the mammalian genome is transcribed as non-coding RNA while only 1–2% of the mammalian genome is transcribed as protein-coding RNA1–3
Composition of the mouse liver transcriptome. 8-week old C57BL6/J male mice were subjected to a standard diet (STD) (n = 6) or cholesterol-rich Western-style high fat diet (HFD) (n = 6)
Exposure of C57BL6/J mice to a 12-week Western-style HFD altered hepatic expression of 395 protein-coding genes, which were statistically overrepresented for functions in steroid and cholesterol metabolism, fatty acid (FA) metabolism, lipid localization, and circadian rhythm

Summary

Introduction

More than 70% of the mammalian genome is transcribed as non-coding RNA (ncRNA) while only 1–2% of the mammalian genome is transcribed as protein-coding RNA1–3. LncRNAs can interact with histone-modifying protein complexes such as the Trithorax group complex and the Polycomb repressive complex 29 or with DNA methyltransferases[10,11] to activate or silence genes. They can alter splicing, editing or stability of protein-coding RNAs12. RNA-seq allows simultaneous quantification of protein-coding and non-coding antisense transcripts originating from complementary DNA strands. We applied RNA-seq to explore the possible existence of cis-regulatory interactions of lncRNAs with protein-coding genes in mouse livers in response to chronic HFD feeding. 5-methylcytosine DNA immunoprecipitation sequencing (5meDIP-seq) and targeted bisulfite sequencing (BS-seq) determined DNA methylation of protein-coding genes as a possible epigenetic endpoint of cis-acting lncRNA/coding gene interactions

Methods

Results

Conclusion