Epigenetic state and gene expression remain stable after CRISPR/Cas-mediated chromosomal inversions

Abstract

Introduction

There is a general correlation between the chromosomal position of a DNA sequence, the epigenetic state of the chromatin as well as gene activity (Grewal & Moazed, 2003; Liu et al., 2016). Chromosome arms are euchromatin-enriched, whereas centromeric and pericentromeric regions are heterochromatic in many species (Roudier et al., 2009). Euchromatin, which is the decondensed fraction of chromatin, contains mostly active genes (Strahl et al., 1999). By contrast, heterochromatin, the condensed chromatin fraction, is poor in genes and gene activity (Fischer et al., 2006; Liu et al., 2016). The formation and maintenance of the chromatin status is regulated epigenetically by DNA methylation and post-translational histone modifications. Heterochromatin is enriched in hypermethylated DNA and dimethylated histone H3K9 (H3K9me2) (Soppe et al., 2002). By contrast, euchromatin is linked with trimethylated H3K4 (H3K4me3) and less C-methylation of DNA.

Position effect variegation (PEV), discovered in the fruit fly Drosophila melanogaster (Gowen & Gay, 1934) and humans (Finelli et al., 2012), as well as the telomere position effect (TPE), discovered in budding yeast, are examples for possible effects of the chromosomal position on gene expression (Gottschling et al., 1990). Genes undergo differential expression in PEV because chromosomal inversions create new heterochromatin–euchromatin borders, and euchromatic genes juxtaposed to heterochromatic regions undergo heterochromatin-induced gene silencing (Hessler, 1958; Elgin & Reuter, 2013). The impact of the chromosomal position on gene expression is well-studied in the case of the expression of the 45S rDNA loci in Arabidopsis thaliana (Mohannath et al., 2016). Also, other studies suggest that changes in gene expression follow the introduction of chromosomal rearrangements, such as inversions or translocations, due to reorganization of large regulatory domains (Naseeb et al., 2016). They are also reported to cause the modification of genetic regions adjacent to the breakpoints (Lavington & Kern, 2017), the epigenetic environment of translocated and adjacent regions (Wesley & Eanes, 1994; Fournier et al., 2010), or to cause nuclear reorganization (Fournier et al., 2010; Harewood et al., 2010). However, it is unknown whether the reported gene expression and epigenetic changes occurred immediately after the introduction of the chromosomal rearrangements or whether they were established over time in subsequent generations.

To unravel the effect of chromosomal inversions on the epigenetic state of chromatin and the activity of genes in A. thaliana, we employed CRISPR/Cas-assisted chromosome engineering for the generation of two differently sized chromosomal inversions (Rönspies et al., 2022a). The inversions were first confirmed by sequencing of the inversion junctions and fluorescent in situ hybridization (FISH). Then, the epigenetic state of these lines was compared with wild-type (WT) plants with the help of whole-genome bisulfite sequencing (WGBS) and chromatin immune precipitation followed by sequencing (ChIP-seq) using antibodies recognizing H3K4me3 and H3K9me2 as eu- and heterochromatic histone marks, respectively. Finally, the effect of the chromosomal rearrangements on the activity of genes was analyzed. Our results showed that none of the studied inverted chromosome segments and their neighboring regions changed in epigenetic marks and gene expression besides minor genome-wide effects, demonstrating the robustness of the epigenome and transcriptome following CRISPR/Cas-induced chromosomal restructuring, at least in the following generations.