Figure 4. Inversion of the CpG-Free Cassette and Analysis of Chromatin Structure of the Inverted Cassette.

(A) Southern blots demonstrating inversion of the 23-β-EGFP cassette from the N to the P orientation. N to P lanes, Hind III digested genomic DNA from three N to P inverted sub-clones isolated after transfection of cells with cassette CpG-free 23-β-EGFP in the N orientation with a Cre recombinase expression plasmid. N and P, control sub-clones.

(B) FACS analysis of representative clones with the CpG-free and CpG-containing cassettes in the P or N orientation or after N to P inversion. x-axis, forward scatter; y-axis, EGFP fluorescence. N to P inversion leads to the reactivation of the CpG-free but not the CpG-containing cassette.

(C) Quantitative RT-PCR analysis of EGFP expression on total RNA extracted from two to five sub-clones with the CpG-containing or the CpG-free cassette in the P or N orientation and after N to P in vivo inversion. Results are expressed as percent of β-2-micro-globulin expression (100 × number of EGFP transcripts/number of B2M transcripts). This analysis confirms the FACS data and demonstrates that silencing and reactivation of the CpG-free cassette occurs primarily at the transcriptional level.

(D) Plots illustrating replication timing analysis of the CpG-free cassette after N to P inversion. The graphs and controls are as in Figure 3C. After N to P inversion, the CpG-free cassette replicates early in S phase.

(E) ChIP analysis of the CpG-free 23-β-EGFP cassette. The graphs and controls are as in Figure 3B. The results show that the CpG-free cassette is enriched in H3 acetylation and poor in H3-K9 tri-methylation in the P orientation, but that it is poor in H3 acetylation and rich in H3-K9 tri-methylation in the N orientation. After N to P inversion, the CpG-free cassette becomes enriched in H3 acetylation but poor in H3-K9 tri-methylation.

N, non-permissive orientation; P, permissive orientation.