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.

Figure 1

PIN*POINT strategy. (a) A diagram of the structure of FokI endonuclease showing the DNA sequence-specific and nuclease domains. (b) The expression vector for Sp1 pointer is cotransfected with a target plasmid that contains the Sp1 binding site into MEL cells. The crescent portion of Sp1 (middle of the diagram) represents the DNA binding domain of Sp1. The flexible linker region and the nuclease domain are represented as a string and an arrowhead, respectively. Low molecular weight DNA is recovered and cleavage in the promoter of the target plasmid is detected by primer extension with a radioactively labeled internal primer (∗). For increased sensitivity, ligation-mediated PCR (LM-PCR) may also be used.

Figure 2

β-globin LCR recruits Sp1 pointer. (a) Diagram of target plasmids p306 (λ) and p269 (L) is shown at the top. The positions of the hypersensitive sites (β-globin LCR) are indicated with vertical arrows. Also shown are the relative positions of the UA region, the tandem CACCC boxes (solid circle), the overlapping GATA-1/CAAT box (solid rectangle), and the TATA box (T) in the β-globin promoter. The transcription initiation site is indicated by a bent arrow.Transcription factor binding sites in the β-globin upstream activator region and the minimal promoter region (15), the positions of the primers (horizontal arrows) used in this article, and the chloramphenicol acetyltransferase reporter gene (CAT) are shown in the enlarged diagram. The distal and proximal CACCC boxes are indicated as CACCC (D) and CACCC (P), respectively. The structure of the Sp1 pointer ABCZD (16) is shown at the bottom. Domains A and B contain serine/threonine- and glutamine-rich regions, interact with TATA binding protein-associated factors, and are required for transcriptional activation and Sp1 tetramer formation. The zinc fingers (Z) bind DNA in a sequence-specific manner, and domain D is thought to mediate Sp1 tetramer-tetramer interaction (–18). (b) Recruitment Sp1 pointer ABCZD to target plasmid p306 (λ) (lanes 1 and 3) or p269 (L) (lanes 2 and 4) was detected with PIN*POINT. Primer extensions with radioactively labeled noncoding-strand primer JS42 (Left) and coding-strand primer JC372 (Right) were performed on the recovered DNA. The positions of the promoter elements near the CACCC boxes are indicated for each strand. Bands 1 and 2 (lanes 2 and 4) correspond to the cleavages 5′ and 3′ of the CACCC boxes, respectively. As shown at the bottom (Recovery), the amount of target plasmid in each sample is similar (19).

Figure 3

Promoter elements participate in the formation of a multimeric Sp1 complex. (a) A diagram of hypothetical Sp1–Sp1 interactions that may recruit the Sp1 pointer (solid oval) lacking the DNA binding domain (ABC) is shown on the left and the structure of the Sp1 pointers ABCD and ABC is shown on the right. (b) Recruitment of Sp1 pointers ABCZD (lanes 1–6), ABCD (lanes 7–12), and ABC (lanes 13–18) to target plasmids containing deletions of different promoter elements was analyzed with PIN*POINT. Primer extension on the noncoding strand is shown. The promoter elements that are present (+) and lacking (−) in each of the target plasmids are summarized in the table below. Positions of the distal CACCC box and TATA box are shown on the right. (c) EMSA analysis was performed with in vitro-translated Sp1 pointers ABCZD and ABC (translated product shown in upper left corner) and oligonucleotide probes (JS72/73, JC416/417, and JC418/419) shown below. The proximal CACCC box and TATA box are in boldface type and the transcription initiation site is marked with a bent arrow. EMSA analysis was done with the probes indicated above and rabbit reticulocyte lysate alone (lanes 1, 5, and 9), in vitro-translated Sp1 pointer ABCZD without (lanes 2, 6, and 10) or with competing Sp1 binding oligonucleotide (lanes 3, 7, and 11), and in vitro-translated Sp1 pointer ABC (lanes 4, 8, and 12). The DNA bound ABCZD complex is indicated with an arrow. Competition with a non-Sp1-binding oligonucleotide did not affect this complex, suggesting that it is a sequence specific complex (data not shown).

Figure 4

LCR hypersensitive sites act synergistically in recruiting Sp1 pointer. Recruitment of Sp1 pointer ABCZD to target plasmids containing various combinations of the 5′HSs of the LCR (lanes: 1, p306; 2, pPN86; 3, p382; 4, p381; 5, p558; 6, p560; 7, p561; 8, p269) shown below was analyzed with PIN*POINT. Primer extension on the noncoding strand is shown. Positions of the UA region, the tandem CACCC boxes (solid circle), the overlapping GATA-1/CAAT box (solid rectangle), and the TATA box (T) of the β-globin promoter are also shown. Primer extension was performed as before (Fig. 2b). The site of the cleavage detectable in lanes 5–8 is approximately 10 bp upstream of the distal CACCC box (see Fig. 2b, band 1). The positions of the distal CACCC box and the TATA box are shown with the size markers.