Fig. 2: Multiple MSR DNA consensus units establish a heterochromatin island and generate bi-directional RNA.

a Diagram of Chr2/116 homology arm DNA constructs having a control DNA sequence (human buffer, HB) or one, three or nine copies of the 234 bp MSR DNA consensus unit. b Genotyping analysis for the validation of correct insertions in two independent homozygous mESC clones, with clone identities indicated by the numbers. c ChIP-qPCR for H3K9me3 with construct-specific T7 and T3 primers in two independent mESC clones for MSR1, MSR3 and MSR9 insertions. Asterisks indicate statistically significant differences when compared to the HB insertion (mean±SD) (****p < 0.0001, ns = not significant, one-way ANOVA, Dunnett’s test). n = 3 independent experiments. d ChIP-qPCR for H3K9me3 with a primer walk into 4 kb 5’ and 3’ flanking regions distal to the Chr2/116 insertion site to examine extension of an H3K9me3 domain. This experiment was done with one mESC clone for MSR1, MSR3 and MSR9 insertions and with WT26 mES cells (mean±SD). n = 3 independent experiments. e RT-qPCR for RNA output in the 5’ and 3’ Chr2/116 homology arms in two independent mESC clones for HB, MSR1 and MSR3 insertions. Expression is normalised to Hprt and is relative to HB control (mean±SD). n = 3 independent experiments. On the right, qPCR cycle 40 products are validated by gel analysis. f ChIP-qPCR for enrichment of (total) RNA polymerase II. Asterisks indicate statistically significant differences compared with the HB control (mean±SD) (*p = 0.0354, **p = 0.0063, ****p < 0.0001, ns = not significant, one-way ANOVA, Dunnett’s test). n = 3 independent experiments.