Fig. 3: LINE 5’UTR repeat units do not establish heterochromatin at the inert genomic region.
From: Transcriptional competence defines the heterochromatin nucleating potential of isolated MSR units
a Diagram of Chr2/116 homology arm DNA constructs having a control DNA sequence (human buffer, HB), three copies of a 208 bp LINE 5’UTR repeat unit (L5’UTR3), three copies of the 228 bp CMV promoter (CMV3) or three copies of the 234 bp MSR consensus unit (MSR3). Genotyping analysis for the validation of correct insertions in two independent homozygous mESC clones, with clone identities indicated by the numbers. b ChIP-qPCR for H3K9me3 (left panel), HP1α (middle panel) and histone H1 (right panel) with construct-specific T7 and T3 primers in two independent mESC clones for HB, L5’UTR3, CMV3 or MSR3 insertions. Asterisks indicate statistically significant differences when compared to the HB control (mean±SD) (**p = 0.0042, ****p < 0.0001, ns = not significant, one-way ANOVA, Dunnett’s test). n = 3 independent experiments. c T7 promoter accessibility assay with one mESC clone for HB, L5’UTR3, CMV3 or MSR3 insertions (left panel), or for HB, MSR1, MSR3 and MSR9 insertions (right panel). Isolated nuclei were incubated with recombinant T7 RNA polymerase and RNA was detected by RT-qPCR with primers specific for the 3’ Chr2/116 homology arm. Asterisks indicate statistically significant differences when compared to the HB control (mean±SD) (****p < 0.0001, one-way ANOVA, Dunnett’s test). n = 3 independent experiments. d Left panel: T7 promoter accessibility assay with one mESC clone for HB, MSR1, MSR3 and MSR9 insertions. Asterisks indicate statistically significant differences when compared to the HB control (mean±SD) (****p < 0.0001, one-way ANOVA, Dunnett’s test). n = 3 independent experiments. Right panel: ChIP-qPCR for histone H1 with construct-specific T7 and T3 primers in one mESC clone for HB, MSR1, MSR3 and MSR9 insertions. Asterisks indicate statistically significant differences when compared to the HB control (mean±SD) (****p < 0.0001, one-way ANOVA, Tukey’s test) or for statistically significant differences between MSR3 and MSR9 insertions (**p = 0.0051, one-way ANOVA, Tukey’s test). n = 3 independent experiments.
