Schematics of the immunoglobulin heavy chain (IgH) locus and of the templates and strategies used for chromatin reconstituted in vitro transcription assays. (A). Eμ within the rearranged VDJ-Cμ murine (IgH) locus. Promoter, p; direction of transcription, rightward arrow; exons, open boxes; 5' and 3' MARs, hatched boxes; Eμ core, filled box. (B). Detailed schematic of the enhancer sites indicating DNA binding sites and proteins that bind to them to activate (positive factors) or to repress (negative factors) transcription (reviewed in ). P1–P4 denote Bright-binding ''P sites'' within the 5' and 3' MARs; the strong P2 site of Bright is indicated by a filled circle. Hinf1 sites (H) operationally define the core region, and XbaI sites (X) flank the MARs. (C). Templates for in vitro chromatin assembly/transcription. VDJ and upstream region was derived from the rearranged VDJ expressed by the BCL1 murine leukemia cell line (37). Templates contain VDJ with no enhancer (P-only) or, ~2 kbp downstream, the Eμ core with (+) or without the 5' and 3' MARs. (D). Probes and protected products used for in vitro transcription reactions and RNAse protection assays. Transcription in vitro and in vitro is initiated from two major start sites (indicated by arrows), resulting in protected products (shown below) of 55 and 43 bases. (E). Order of addition and strategy for in vitro transcription reactions. As detailed in the text, Step 1 conditions examine transcription following addition of factors (extract only, recombinant Bright, or Bright-supplemented extract) or buffer alone prior to chromatin assembly upon templates. Step 2 conditions measure transcription when factors are added after assembly of chromatin. Transcription is initiated by addition of nucleoside-triphosphates (NTPs).