TSA treatment activates the endogenous κ gene. (A) Map of the κ locus showing the promoters (arrows) of the 1.1-kb (5′) and 0.8-kb (3′) germ-line transcripts, the positions of Jκ1–5, and the constant region (Cκ). Shown is RT-PCR analysis of p53^–/– and p53^–/–/Dnmt1^–/– fibroblasts for the 0.8- and 1.1-kb Igκ germ-line (GLκ) transcripts with (+) or without (–) TSA treatment. The mouse Aprt gene is used as a control for these experiments. (B) p53^–/– or p53^–/–/Dnmt1^–/– cells with or without transfected Rag expression vectors were treated with TSA and assayed by RT-PCR for Rag1 and Rag2 expression using β-actin as a control. LM-PCR (see diagram) was used to detect DSB at the Jκ1 RSS using α-actin as a control for DNA content. Southern blot analysis using four different methyl-sensitive restriction enzymes showed that the κ gene region is unmethylated in Dnmt1^– cells (data not shown).

DNaseI sensitivity of the methylated, unmethylated, and demethylated EGFP reporter genes. (A) Cells from methylated EGFP-containing clones were mixed with cells from unmethylated or demethylated clones before nuclei preparation and subsequent DNaseI treatment. The digested DNA was further cut with EcoRI/SacI and HhaI, to distinguish between methylated and unmethylated EGFP molecules, and subjected to blot hybridization using the radioactive probe indicated in Fig. 1 A. (B) The graph (based on phosphorimaging of the blot) shows EGFP reporter gene DNaseI sensitivity of methylated, unmethylated, and demethylated clones. Similar results were obtained by using pools of methylated or demethylated clones (data not shown and Table 2).

TSA treatment activates rearrangement and transcription of the demethylated EGFP reporter gene. (A) Genomic DNA was prepared from um-cl1, dm-cl2, and m-cl3 cells that were treated with or without TSA (10 ng/ml for 24 h), and then amplified with nested primers (1;3 and 2;4, see Fig. 2) that detect rearranged (Re) molecules. α-Actin was used as a control for measuring the amount of input DNA. Similar results were obtained for other unmethylated (4), demethylated (6), and methylated (11) clones (Table 2). (B) RT-PCR analysis of EGFP transcription in unmethylated, demethylated, and methylated clones with (+) or without (–) TSA treatment. Amplification was done with nested primers (1;8 and 2;7, see Fig. 2). The β-actin gene was used as a control for these experiments. Similar results were obtained for other unmethylated (4), demethylated (6), and methylated (5) clones (Table 2). (C) dm-cl2 was single-cell cloned to obtain dm-cl2′. Treated dm-cl2′ cells were grown in the absence of TSA, subcloned, and then examined for EGFP transcription by RT-PCR as above.

DNA methylation represses EGFP reporter gene rearrangement. A schematic diagram of EGFP reporter gene rearrangement is shown. The arrows represent the locations of primers. Genomic DNA from um-cl1, dm-cl2, and m-cl3 was amplified by a first set of primers (1;3) and then by a nested set of primers (2;4) to detect rearrangement. The α-actin gene was used as a control for the amount of input DNA. Similar results were obtained for all of the methylated, unmethylated, and demethylated clones used in this study.

Methylation status of the integrated EGFP reporter gene. (A) A map of the EGFP recombination reporter gene. EGFP (speckled line) is in inverse orientation with regard to the 5′ LTR (dark line), flanked by 12 RSS (open triangle) and 23 RSS (filled triangle). Restriction enzyme sites for EcoRI (R), HpaII (Hp), HhaI (Hh), NcoI (N), and SacI (S) are marked. The line below the restriction map represents the EcoRI–NcoI fragment used as a radioactive probe. (B) Methylation pattern of the transfected EGFP reporter gene. The EGFP reporter gene was methylated in vitro by using HhaI and HpaII methylases, and both the modified and unmodified constructs were cotransfected with a plasmid carrying the neo^r selection gene into 38B9 cells. Neomycin-resistant colonies were expanded and then used to prepare DNA for Southern blot analysis. DNA from unmethylated (um), methylated (m), and demethylated (dm) clones (cl) was digested with NcoI/EcoRI with or without HhaI and HpaII and was subjected to blot hybridization using the EGFP radioactive probe (A). Similar results were obtained from all of the unmethylated (26), methylated (25), and demethylated (7) clones used in this study. Although this analysis examines only the methylation state of a subset of sites within the EGFP regions, all of the HpaII and HhaI sites shown in the figure were assayed by methyl-sensitive PCR and shown to be <10% methylated in the demethylated clones.

Histone modification of EGFP reporter genes. Micrococcal nuclease-prepared mononucleosomes from individual clones um-cl1, dm-cl2, and m-cl3 or from pools (pl) of unmethylated (um), demethylated (dm), and methylated (m) clones were precipitated with antibodies directed against acetylated histone H4 (A) or methylated histone H3(K9) (B). DNA was extracted from unbound and bound fractions and subjected to quantitative PCR using primers (6;7) (see Fig. 2) for the EGFP region and for mouse β- and α-actin as controls. Three concentrations (1-, 3-, and 9-fold) are shown. ChIP of Ac-H4 was also carried out on pooled colonies (data not shown). (C) The data in A and B have been redrawn in graphic form. For experiments on Ac-H4, the degree of enrichment (bound/unbound) is normalized to that obtained for the α-actin gene in each cell type, whereas experiments on Me-H3(K9) were normalized to that obtained for the β-actin gene. It should be noted that identical results were obtained for the LTR region of the vector.