Fig. 1: Intact, but not permutated, MSR units induce H3K9me3 at an inert genomic region.
From: Transcriptional competence defines the heterochromatin nucleating potential of isolated MSR units
a UCSC genome browser shot of the intergenic region in mouse chromosome 2 at position 116 Mbp (Chr2/116 insertion site) that has a 20 kb repeat- and gene-free window. b Diagram of Chr2/116 homology arm DNA constructs having a control DNA sequence (human buffer, HB) (750 bp) or one or three copies of the per-3/99 (300 bp) or the act-9/35 (261 bp) MSR variant units. In the diagrams for the MSR variants, the red lines illustrate point mutations as compared to the MSR DNA consensus sequence. These constructs were inserted into the Chr2/116 integration site by hit-and-run CRISPR/Cas9 technology. Genotyping analysis for the validation of correct Chr2/116 insertions for per-3/99 and act-9/35 MSR variant copies in homozygous mESC clones. c Left panel: ChIP-qPCR for H3K9me3 with construct-specific T7 and T3 primers across the per-3/99 and act-9/35 MSR insertions. Asterisks indicate statistically significant differences compared with the HB control (mean±SD) (****p < 0.0001, one-way ANOVA, Dunnett’s test). n = 3 independent experiments. Right panel: RT-qPCR for RNA output in the 5’ and 3’ Chr2/116 homology arms of per-3/99 and act-9/35 MSR insertions. Expression is normalised to Hprt and is relative to HB control (mean±SD). (****p < 0.0001, one-way ANOVA, Dunnett’s test). n = 3 independent experiments.
