Studying the recruitment of Sp1 to the beta-globin promoter with an in vivo method: protein position identification with nuclease tail (PIN*POINT).

Abstract

Transcription is thought to be regulated by recruitment of transcription factors, adaptors, and certain enzymes to cis-acting elements through protein-DNA interactions and protein-protein interactions. To better understand transcription, a method with the capability to detect in vivo recruitment of these individual proteins will be essential. Toward this end, we use a previously undescribed in vivo method that we term protein position identification with nuclease tail (PIN*POINT). In this 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 cleavage near the binding site. In this article, we used the technique protein position identification with nuclease tail to study the effect of the beta-globin locus control region (LCR) and promoter elements on the recruitment of transcription factor Sp1 to the beta-globin promoter. We present evidence that the hypersensitive sites of the LCR synergistically enhance the recruitment of a multimeric Sp1 complex to the beta-globin promoter and that this may be accomplished by protein-protein interactions with proteins bound to the LCR, the upstream activator region, and, possibly, general transcription factors bound near the "TATA" box.