DISSECTING A LOCUS-CONTROL REGION - FACILITATION OF ENHANCER FUNCTIONBY EXTENDED ENHANCER-FLANKING SEQUENCES

Using transgenic mice, we have defined novel gene regulatory elements, termed ''facilitators.'' These elements bilaterally flank, by up to 1 kb, a 200-bp T-cell-specific enhancer domain in the human adenosine d eaminase (ADA) gene, Facilitators were essential for gene copy-proport ional and integration site-independent reporter expression in transgen ic thymocytes, but they had no effect on the enhancer in transfected T cells. Both segments were required. Individual segments had no activi ty. A lack of facilitator function caused positional susceptibility an d prevented DNase I-hypersensitive site formation at the enhancer. The segments were required to be at opposed ends of the enhancer, and the y could not be grouped together. Reversing the orientation of a facili tator segment caused a partial loss of function, suggesting involvemen t of a stereospecific chromatin structure. trans-acting fatter access to enhancer elements was modeled by exposing nuclei to a restriction e ndonuclease. The enhancer domain was accessible to the 4-cutter DpnII in a tissue- and cell-type-specific fashion. However, unlike DNase I h ypersensitivity and gene expression, accessibility to the endonuclease could occur without the facilitator segments, suggesting that an acce ssible chromatin domain is an intermediate state in the activational p athway. These results suggest that facilitators (i) are distinct from yet positionally constrained to the enhancer, (ii) participate in a ch romatin structure transition that is necessary for the DNase I hyperse nsitivity and the transcriptional activating function of the enhancer, and (iii) act after cell-type-specific accessibility to the enhancer sequences is established by factors that do not require the facilitato rs to be present.