STUDYING THE RECRUITMENT OF SP1 TO THE BETA-GLOBIN PROMOTER AN IN-VIVO METHOD - PROTEIN POSITION IDENTIFICATION WITH NUCLEASE TAIL (PIN-ASTERISK-POINT)

Transcription is thought to be regulated by recruitment of transcripti on factors, adaptors, and certain enzymes to cis-acting elements throu gh protein-DNA interactions and protein-protein interactions, To bette r understand transcription, a method with the capability to detect in vivo recruitment of these individual proteins will be essential, Towar d this end, we use a previously undescribed in vivo method that we ter m protein position identification with nuclease tail (PINPOINT). In t his method, a fusion protein composed of a chosen protein linked to a nonsequence-specific nuclease is expressed in vivo, and the binding of the protein to DNA is made detectable by the nuclease induced cleavag e near the binding site, In this article, we used the technique protei n position identification with nuclease tail to study the effect of th e beta-globin locus control region (LCR) and promoter elements on the recruitment of transcription factor Spl to the beta-globin promoter, W e present evidence that the hypersensitive sites of the LCR synergisti cally enhance the recruitment of a multimeric Spl complex to the beta- globin promoter and that this may be accomplished by protein-protein i nteractions with proteins bound to the LCR, the upstream activator reg ion, and, possibly, general transcription factors bound near the ''TAT A'' box.