3D disorganization and rearrangement of genome provide insights into pathogenesis of NAFLD by integrated Hi-C, Nanopore, and RNA sequencing

Abstract

The three-dimensional (3D) conformation of chromatin is integral to the precise regulation of gene expression. The 3D genome and genomic variations in non-alcoholic fatty liver disease (NAFLD) are largely unknown, despite their key roles in cellular function and physiological processes. High-throughput chromosome conformation capture (Hi-C), Nanopore sequencing, and RNA-sequencing (RNA-seq) assays were performed on the liver of normal and NAFLD mice. A high-resolution 3D chromatin interaction map was generated to examine different 3D genome hierarchies including A/B compartments, topologically associated domains (TADs), and chromatin loops by Hi-C, and whole genome sequencing identifying structural variations (SVs) and copy number variations (CNVs) by Nanopore sequencing. We identified variations in thousands of regions across the genome with respect to 3D chromatin organization and genomic rearrangements, between normal and NAFLD mice, and revealed gene dysregulation frequently accompanied by these variations. Candidate target genes were identified in NAFLD, impacted by genetic rearrangements and spatial organization disruption. Our data provide a high-resolution 3D genome interaction resource for NAFLD investigations, revealed the relationship among genetic rearrangements, spatial organization disruption, and gene regulation, and identified candidate genes associated with these variations implicated in the pathogenesis of NAFLD. The newly findings offer insights into novel mechanisms of NAFLD pathogenesis and can provide a new conceptual framework for NAFLD therapy.

Keywords: 3C, chromosome conformation capture; 3D genome; 3D, three-dimensional; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Abcg5, ATP-binding cassette sub-family G member 5; BWA, Burrows-Wheeler Aligner; CNV, copy number variation; Camk1d, calcium/calmodulin-dependent protein kinase type 1D; Chr, chromosome; Chromatin looping; DEG, differentially expressed gene; DEL, deletion; DI, directionality index; DUP, duplication; Elovl6, elongation of very long chain fatty acids protein 6; FDR, false discovery rate; FFA, free fatty acid; Fgfr2, fibroblast growth factor receptor 2; GCKR, glucokinase regulator; GO, gene ontology; GSH, glutathione; Gadd45g, growth arrest and DNA damage-inducible protein GADD45 gamma; Grm8, metabotropic glutamate receptor 8; Gsta1, glutathione S-transferase A1; H&E, hematoxylin-eosin; HFD, high-fat diet; HSD17B13, hydroxysteroid 17-beta dehydrogenase 13; Hi-C, high-throughput chromosome conformation capture; IDE, interaction decay exponent; INS, insertion; INV, inversion; IR, inclusion ratio; IRGM, immunity related GTPase M; IRS4, insulin receptor substrate 4; KEGG, Kyoto Encyclopedia of Genes and Genomes; Kcnma1, calcium-activated potassium channel subunit alpha-1; LPIN1, lipin 1; MBOAT7, membrane bound O-acyltransferase domain containing 7; MDA, malondialdehyde; NAFLD, non-alcoholic fatty liver disease; NF1, neurofibromin 1; NGS, next-generation sequencing; NOTCH1, notch receptor 1; ONT, Oxford Nanopore Technologies; PCA, principal component analysis; PNPLA3, patatin like phospholipase domain containing 3; PPP1R3B, protein phosphatase 1 regulatory subunit 3B; PTEN, phosphatase and tensin homolog; Pde4b, phosphodiesterase 4B; Plce1, 1-phosphat-idylinositol 4,5-bisphosphate phosphodiesterase epsilon-1; Plxnb1, Plexin-B1; RB1, RB transcriptional corepressor 1; RNA-seq, RNA-sequencing; SD, standard deviation; SOD, superoxide dismutase; SV, structural variation; Scd1, acyl-CoA desaturase 1; Sugct, succinate-hydroxymethylglutarate CoA-transferase; TAD, topologically associated domain; TC, total cholesterol; TG, triglyceride; TM6SF2, transmembrane 6 superfamily member 2; TP53, tumor protein p53; TRA, translocation; Topologically associated domain; Transcriptome; WGS, whole-genome sequencing; Whole-genome sequencing.