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

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 β-globin locus control region (LCR) and promoter elements on the recruitment of transcription factor Sp1 to the β-globin promoter. We present evidence that the hypersensitive sites of the LCR synergistically enhance the recruitment of a multimeric Sp1 complex to the β-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.