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

Abstract

The epigenetic state of chromatin, gene activity and chromosomal positions are interrelated in plants. In Arabidopsis thaliana, chromosome arms are DNA-hypomethylated and enriched with the euchromatin-specific histone mark H3K4me3, while pericentromeric regions are DNA-hypermethylated and enriched with the heterochromatin-specific mark H3K9me2. We aimed to investigate how the chromosomal location affects epigenetic stability and gene expression by chromosome engineering. Two chromosomal inversions of different sizes were induced using CRISPR/Cas9 to move heterochromatic, pericentric sequences into euchromatic regions. The epigenetic status of these lines was investigated using whole-genome bisulfite sequencing and chromatin immunoprecipitation. Gene expression changes following the induction of the chromosomal inversions were studied via transcriptome analysis. Both inversions had a minimal impact on the global distribution of histone marks and DNA methylation patterns, although minor epigenetic changes were observed across the genome. Notably, the inverted chromosomal regions and their borders retained their original epigenetic profiles. Gene expression analysis showed that only 0.5-1% of genes were differentially expressed genome-wide following the induction of the inversions. CRISPR/Cas-induced chromosomal inversions minimally affect epigenetic landscape and gene expression, preserving their profiles in subsequent generations.

Keywords: CRISPR/Cas; chromosome engineering; epigenetics; gene expression; inversion.

Fig. 1

Generated and analyzed CRISPR‐SaCas9‐induced Arabidopsis thaliana inversion lines. (a) Line CS1282 with a re‐inversion of the hk4S knob region on chromosome IV (Schmidt et al., 2020), (b) line RW290 with a 5‐Mb‐large paracentric and (c) line RW295 with a 7.5‐Mb‐large pericentric inversion of chromosome III. Positions of the inversion breakpoints and applied fluorescent in situ hybridization (FISH) probes are indicated. FISH of wild‐type (WT) A. thaliana Col‐0 (d) mitotic and (e) pachytene chromosomes with the chromosome III‐specific probes 1 (magenta) and 2 (green) and the centromere‐specific probe 3 (yellow). (f) FISH of line RW290 pachytene chromosomes with the chromosome III‐specific probes 1, 2 and the centromere‐specific probe. Compared with the WT, the green signal moved away from the centromere signal in the inversion line. Instead, the magenta signal moved into the vicinity of the yellow signal. (g) FISH of line RW295 pachytene chromosomes with the chromosome III‐specific probe 1 and the centromere‐specific probe 3. Compared with the WT, the green signal moved away from the centromere signal in the inversion line. Chromatin was counterstained with DAPI. Insets show chromosome III further enlarged. The magenta, green and yellow arrows show the position of probes 1, 2 and 3 signals, respectively.

Fig. 2

Global distribution of histone marks specific to eu‐ and heterochromatin remains unaltered following the induction of chromosomal inversions. (a) Similar genome‐wide distribution of eu‐ (H3K4me3) and heterochromatic (H3K9me2) histone marks between lines RW290, RW295, CS1282 and wild‐type (WT) Arabidopsis thaliana Col‐0. (b) Further resolved distribution of H3K4me3 and H3K9me2 marks within the inversion segments and proximal to the breakpoints (±100 kb). To allow visual comparison of epimarks along the chromosomes, the inverted chromosome segments of all three lines are shown in an inverted orientation. The comparisons are not in scale. Note, Supporting Information Fig. S3 shows the plotted data against a physically rearranged genome assembly.

Fig. 3

Global DNA methylome remains preserved following the induction of the chromosome segment inversions. (a) Global DNA methylation pattern of all C contexts over all chromosomes of the Arabidopsis thaliana lines carrying an inversion compared with the wild‐type (WT). (b) Comparison of different C context methylation levels compared with WT Col‐0 in the area of the inversion and the ±100 kb flanking regions. The dotted blue line indicates the breakpoint positions. To allow visual comparison of DNA methylation marks along the chromosomes, the inverted chromosome segments of all three lines are shown in an inverted orientation.

Fig. 4

Gene expression changed to some extent after the induction of chromosome segment inversions in Arabidopsis thaliana. (a) More than 1000 differentially expressed genes (DEGs) were identified in each inversion line. (b) From the total number of DEGs, a modest number of DEGs, specifically 139, 167 and 327 genes were recognized to be specific to lines RW290, CS1282 and RW295, respectively. A total of 1297 DEGs were shared in all inversion lines. (c) Gene expression profile of RW290, CS1282 and RW295 in the inverted and flanking regions to the break points. The expression profile of each line compared to the control was not affected by the inversion events in the inversion segments and the ±100 kb flanking regions. To allow visual comparison of DEGs along the chromosomes, the inverted chromosome segments of all three lines are shown in an inverted orientation.

Fig. 5

Model of the effect of a chromosome segment inversion on gene expression. Despite the formation of new eu/heterochromatic borders in the inversion lines, the genes located near the inversion borders mostly did not alter their expression due to the juxtaposition to eu‐ or heterochromatin except for a few genes. The activity and position of the genes are shown as red (downregulated), blue (upregulated) and white (not affected) points.