Histone hyperacetylation is insufficient for recombinational accessibility. Acetylation levels of histones H3 and H4 associated with the Dβ1 and Jβ1.1 gene segments are shown for independent pools of 5B3 transfectants (Exp. 1 and Exp. 2). Chromatin immunoprecipitation assays were performed on mononucleosome preparations with antibodies specific for acetylated lysines on H3 (black bars) or H4 (white bars). Acetylation of histones at the endogenous TEA element, which is inactive in 5B3 cells, is shown as a negative control. Acetylation values were calculated as described in Materials and Methods.

Promoters and enhancers confer RAG accessibility to RSSs. (A) Schematic depiction of TCRβ miniloci. NotI (N) and XhoI (X) sites used to introduce PDβ and iEκ are shown. (B) Generation of SEs from chromosomal TCRβ miniloci. Genomic DNA was harvested from independent pools of 5B3 transfectants (letters above each lane) containing the specified miniloci (lanes 3–14) or untransfected 5B3 (lanes 1 and 2). All pooled transfectants harbored an average of 5–6 substrate copies and are derived from ≥20 clones. Cells were cultured in the absence or presence of tetracycline (24 h) and subjected to LM-PCR analysis (13) to measure induced cleavage of the 3′Dβ RSS. Control assays for recombinase activity (endogenous Jλ2/3-SEs) and DNA content (Cλ) are shown in Middleand Bottom, respectively. The linearity of each assay was confirmed by serial dilution (lanes 15–18) of the RAG-induced P⁺E⁺ sample shown in lane 12. Similar results were obtained with multiple independent subclones for each construct (data not shown).

Restriction endonuclease sensitivity is independent of promoter location. (A) Diagram of LM-PCR strategies for XmnI or EcoRV cleavage products. BW-1/2 linkers are shown in bold, and TCRβ-specific primers are indicated by arrows. (B and E) Levels of RE cleavage products within modified substrates. Nuclei from 5B3 clones containing the indicated TCRβ substrate or untransfected 5B3 cells were incubated with XmnI (0, 0.5, 1, 5, and 10 units) or EcoRV (at 0, 0.1, 0.5, 1, and 5 units). Genomic DNA from treated nuclei was subjected to nested LM-PCR for cleavage products (A). The linearity of each assay was confirmed by serial dilutions (lanes 21–24) of the maximally digested P⁺E⁺ sample (lane 15). (C, D, F, and G) Quantification of RE sensitivity in TCRβ miniloci. LM-PCR signals for XmnI (5 units) or EcoRV (5 units) treatment were quantified by PhosphorImager analysis. Values were normalized to signals obtained with PCR assays for DNA content (Cλ), substrate copy number (Jβ1.1), and cleavage of accessible loci (Cκ for XmnI or c-myc for EcoRV). Normalized values are shown relative to data for P⁺E⁺ (C and E) or P-DJ (D and F).

Promoter positioning affects substrate rearrangement independent of germ-line transcription. (A) Diagram of iEκ-containing miniloci harboring repositioned promoters. A PDβ cassette was introduced into the TCRβ minilocus in both orientations (P and P′) at the indicated sites. Structures of JβCμ and Iμ transcripts, as well as the reverse transcription–PCR primers used to detect JβCμ germ-line transcripts (primers A and B) and DβJβ rearrangement (primers C and D) are shown. (B) Germ-line transcription in miniloci with repositioned promoters. Total RNA from independent pools of 5B3 transfectants (letters above each lane) containing the specified miniloci were subjected to a reverse transcription–PCR assay specific for JβCμ transcripts (A). Total cDNA levels were measured by using a PCR assay for β-actin transcripts (Lower). The linearity of each assay was confirmed by serial dilution of the P-DJ sample shown in lane 6 (lanes 12–15). Similar results were obtained with multiple independent subclones for each construct and with an assay that measures spliced transcripts originating within the Dβ RSS (data not shown). (C) DβJβ rearrangement in TCRβ miniloci. Genomic DNA was harvested from independent pools of 5B3 transfectants for each substrate (letters above each lane). Pooled cells were cultured in either the absence or presence of RAG expression (48 h). PCR assays (A) yielded amplification products corresponding to Dβ1Jβ1.1 (DJβ1) or Dβ1Jβ1.2 (DJβ2) rearrangements (Top). Control assays for recombinase activity (endogenous VλJλ rearrangement) and total DNA content (Cλ) are shown in Middle and Bottom, respectively. The linearity of each assay was confirmed by serial dilution of the RAG-induced P-DJ sample shown in lane 10 (lanes 19–22). Similar results were obtained with multiple independent subclones for each construct (data not shown).