Chromosome Conformation of Human Fibroblasts Grown in 3-Dimensional Spheroids

Abstract

In the study of interphase chromosome organization, genome-wide chromosome conformation capture (Hi-C) maps are often generated using 2-dimensional (2D) monolayer cultures. These 2D cells have morphological deviations from cells that exist in 3-dimensional (3D) tissues in vivo, and may not maintain the same chromosome conformation. We used Hi-C maps to test the extent of differences in chromosome conformation between human fibroblasts grown in 2D cultures and those grown in 3D spheroids. Significant differences in chromosome conformation were found between 2D cells and those grown in spheroids. Intra-chromosomal interactions were generally increased in spheroid cells, with a few exceptions, while inter-chromosomal interactions were generally decreased. Overall, chromosomes located closer to the nuclear periphery had increased intra-chromosomal contacts in spheroid cells, while those located more centrally had decreased interactions. This study highlights the necessity to conduct studies on the topography of the interphase nucleus under conditions that mimic an in vivo environment.

Chromosome conformation and gene expression patterns differ profoundly in human fibroblasts grown in spheroids versus monolayers

“…It has previously been shown that chromosome 18 had increased intra-chromosomal interactions while the chromosomes whose TADs changed the most, including 19, had decreased intrachromosomal interactions. 13 Additionally, chromosome 19 has the highest gene density while chromosome 18 has the lowest, which further suggests that gene expression and chromosomal structure are tightly coupled. Fig.…”

“…After 48-h of growth, we sampled the cells for Hi-C and RNA-seq analyses. Hi-C libraries were constructed from 20 million cells for each culturing condition as described by Chen et al 13 Briefly, we used the HindIII restriction enzyme (RE) for chromatin digestion. RE created DNA fragment ends were marked with biotin-dCTP (Cat# 19518-018, Life Technologies) and re-ligated.…”

“…We previously observed chromosome conformation changes between human fibroblasts grown as spheroids vs. monolayer cultures. 13 Here we extend our investigation into how genome conformation (structure) changes affect changes in genomewide transcription (function). We focus on the nucleome of human fibroblasts grown in 3D and 2D cultures for 48 hours.…”

Nucleome Analysis Reveals Structure–Function Relationships for Colon Cancer

“…Hi-C, RNA-seq, and FISH data were collected from human fibroblasts and HT-29 cell lines as described by Chen and colleagues (12). Cell culture in 2D and three dimensional (3D) growth was performed as described by Chen and colleagues (13). Extended protocols for RNA-seq, Hi-C, and FISH are mentioned in the Supplementary Methods.…”

“…18) with official gene symbols and the default background set for human analysis. The statistical test comparing samples was performed as described by Chen and colleagues (13).…”