Studying the recruitment of Sp1 to the beta-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 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.
Lee
J S
JS
Molecular Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive, MSC 1654, Bethesda, MD 20892-1654, USA.
Lee
C H
CH
Chung
J H
JH
D001483:Base Sequence; D002460:Cell Line; D018983:DNA Footprinting; D005914:Globins / Q000235:genetics*; D006801:Humans; D019901:Locus Control Region / Q000502:physiology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D016385:TATA Box; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection
D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D005914:Globins