Figure 3. Analysis of the Chromatin Structure of the CpG-Containing Cassette.

(A) Bisulfite sequencing analysis was performed on cells with CpG-containing cassette 234-β-EGFP at RL4 after N to P inversion. There were 11 molecules sequenced. Most of the CpGs were methylated (see text).

(B) Histograms illustrating H3 acetylation and H3-K4 methylation results on the CpG-containing cassettes. The analysis was performed as in Figure 2. Mouse β-major globin (mBmaj) is a positive control for H3 and H4 acetylation and a negative control for H3-K9 tri-methylation. The α subunit of the guanine nucleotide-binding protein (mGnasx) and the mouse amylase genes are a positive control for H3-K9 tri-methylation and negative controls for H3 acetylation. The CpG-containing cassette is enriched in H3 acetylation and H3-K4 methylation in the P orientation but poor in acetylated H3 and H3-K4 methylation. After N to P inversion, the cassette remains poor in H3 acetylation and H3-K4 methylation.

(C) Plots illustrating replication timing analysis of the CpG-containing cassette after N to P inversion. The analysis was performed as in Figure 2. Mouse β-major globin and amylase are early and late replicating controls. After N to P inversion, the CpG-containing cassette replicate late in S phase.

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

(D) Histograms illustrating H3-K9 tri-methylation results on the CpG-containing cassettes. The analysis was performed as in Figure 2. The controls are as in Figure 3B. The enrichment is low when the cassette is in the P orientation and high in the N orientation. The enrichment remains high after N to P inversion.