Activation of beta-globin promoter by erythroid Krüppel-like factor

Abstract

Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the beta-globin gene CACCC box and is essential for beta-globin gene expression. EKLF does not activate the gamma gene, the CACCC sequence of which differs from that of the beta gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the beta gene by EKLF, the CACCC boxes of beta and gamma genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the beta promoter carrying a gamma CACCC box at a level comparable to that at which it activated the wild-type beta promoter, whereas EKLF failed to activate a gamma promoter carrying the beta CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the beta promoter carrying the gamma CACCC box, or that EKLF activated the mutated beta promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the beta or gamma promoter determined EKLF specificity, the proximal beta CACCC box sequence was created at the position of the beta promoter (-140) which corresponds to the position of the CACCC box on the gamma promoter. Similarly, the beta CACCC box was created in the position of the gamma promoter (-90) corresponding to the position of the CACCC box in the beta promoter. EKLF retained weak activation potential on the beta(-140CAC) promoter, whereas EKLF failed to activate the gamma(-90betaCAC) promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the beta promoter by EKLF is determined by the overall structure of the beta promoter rather than solely by the sequence of the beta gene CACCC box.

FIG. 1

(A) Structure of pHS2β^γCACCAT, containing a β gene promoter with a γ CACCC box. The CAT gene is driven by a 1.5-kb KpnI-BglII fragment of HS2 and the β gene promoter (a fragment extending from bp −265 to +48 relative to the cap site) carrying a γ CACCC box. Uppercase letters denote the 9-bp γ CACCC sequence analogous to the β CACCC sequence recognized by EKLF. Numbers above the γ CACCC box sequence show base pair distances from the cap site. (B) Transactivation of a β promoter containing a γ CACCC box by EKLF. CAT activities in CV-1 cells were normalized to β-Gal activity and expressed as relative percentages of CAT activity of pHS2βCAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are expressed as mean (columns) ± SD (error bars) derived from four independent transfections using two different plasmid sets. Notice that EKLF transactivates the β gene promoter despite the fact that the promoter contains the γ CACCC box.

FIG. 2

(A) Structure of the construct pHS2γ^βCACLuc containing a γ gene promoter with a β CACCC box. The luciferase gene is driven by the 1.5-kb fragment of HS2 and the γ gene promoter (bp −299 to +37 relative to the cap site) carrying a β CACCC box. Uppercase letters denote the 9-bp β sequence recognized by EKLF. Numbers above the β CACCC box sequence show base pair distances from the cap site. (B) Transactivation of a γ promoter containing a β CACCC box by EKLF. Luciferase activities in CV-1 cells were normalized to β-Gal activity and expressed as relative percentages of luciferase activity of pHS2γLuc in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that EKLF cannot transactivate the γ gene promoter despite the fact that the promoter contains the β CACCC box.

FIG. 3

Studies using K562 cells. Results of transactivation by EKLF of a β promoter carrying a γ CACCC box and a γ promoter carrying a β CACCC box are depicted. CAT and luciferase activities were normalized to β-Gal activity and expressed as relative percentages of CAT and luciferase activities of pHS2βCAT and pHS2γLuc in K562 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that the CACCC box substitutions do not influence the level of activation of the β or γ gene promoter by EKLF.

FIG. 4

Relationship between amounts of transfected activator plasmid and degree of activation of the reporter gene in CV-1 cells. The reporter construct, pHS2βCAT, was cotransfected with various amounts of pSG5/EKLF. CAT activities were normalized to β-Gal activity and expressed as relative percentages of CAT activity of pHS2βCAT transfected with 3.6 μg of pSG5/EKLF (100%). Average values ± SD (error bars) were derived from three independent transfections using two different plasmid sets. Notice that CAT activities show two phases, ascending (0 to 0.7 μg of EKLF plasmid) and plateau (0.7 to 3.6 μg of EKLF plasmid).

FIG. 5

Transactivation of the β promoter containing a γ CACCC box by a small amount of pSG5/EKLF (0.5 μg per transfection). CAT activities in CV-1 cells were normalized to β-Gal activity and expressed as relative percentages of CAT activity of pHS2βCAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from three independent transfections using two different plasmid sets. Notice that EKLF transactivates the β gene promoter containing the γ. CACCC box, a result which is similar to the results of assays using a standard amount (3.6 μg) of pSG5/EKLF (Fig. 1).

FIG. 6

(A) Disruption of the distal CACCC box sequence (β^γCACΔdCAC). The distal CACCC box sequence (CCT CAC CCT) of the β promoter carrying a γ CACCC box sequence (CTC CAC CCA) at the proximal CACCC site is altered to CCT ACT AGT. Numbers above the promoter sequences show base pair distances from the cap site. (B) Transactivation of a distal CACCC box-disrupted β^γCAC promoter by EKLF. A small amount of EKLF plasmid (0.5 μg) was used for transfection. CAT activities in CV-1 cells were normalized to β-Gal activity and expressed as relative percentages of CAT activity of pHS2β^γCACCAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from three independent transfections using two different plasmid sets. Notice that in the presence of EKLF, similar levels of CAT activities were obtained from β^γCAC and β^γCACΔdCAC.

FIG. 7

Comparison of the locations of cis elements of the β and γ gene promoters. The positions of the functional CACCC box are shown by solid rectangles.

FIG. 8

Effect of the position of the CACCC box on activation of the γ gene promoter by EKLF. (A) Generation of a γ promoter containing a β CACCC box at bp −90 (γ^−90βCAC). The original γ CACCC box sequence (CTC CAC CCA) was deleted, and the proximal β CACCC box sequence was inserted into position −90, i.e., the position of the β CACCC box in the β promoter. Numbers above the promoter sequences are base pair distances from the cap site. (B) Transactivation of a γ promoter containing a β CACCC box at position −90 (β^−90βCAC) by EKLF. Luciferase activities in CV-1 cells were normalized to β-Gal activity and expressed as relative percentages of luciferase activity of pHS2γLuc in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that the promoter activity is almost ablated by the CACCC box movement. In addition, EKLF cannot activate the mutant promoter even though this promoter contains an optimal binding sequence at its optimal site.

FIG. 9

Effect of the position of the CACCC box on the activation of the β-globin gene promoter by EKLF. (A) Generation of a β promoter containing a CACCC box at bp −140 (β^−140CAC), i.e., the position of the CACCC box in the γ promoter. The proximal CACCC box sequence (CCA CAC CCT) and the distal CACCC box sequence (CCT CAC CCT) were disrupted by base substitutions (shown in underlined italics). Subsequently, the proximal CACCC box sequence was inserted into the γ CACCC position in the γ promoter. Numbers above the promoter sequences correspond to base pair distances from the cap site. (B) Transactivation of a β promoter containing a CACCC box at position −140 (β^−140CAC) by EKLF. CAT activities in CV-1 cells were normalized to β-Gal activity and expressed as relative percentages of CAT activity of pHS2βCAT in CV-1 cells which were not transfected by a transactivator plasmid (100%). Data are derived from four independent transfections using two different plasmid sets. Notice that the promoter activity is remarkably decreased by the relocation of the CACCC box to a position equivalent to that of the CACCC box of the γ gene and that EKLF is still capable of weakly activating this mutant promoter.

FIG. 10

Proposed mechanism of β-globin gene activation by EKLF. (A) EKLF bound to the β gene recruits a subcomplex of the transcriptional machinery and enables formation of a transcription initiation complex (IC) of the β gene together with TFIID containing TATA box-binding protein (TBP), RNA polymerase II (pol II) holoenzyme, and probably other subcomplexes recruited by other transcriptional activators. This initiation complex gives rise to high-level β gene transcription. (B) EKLF tethered to the γ gene by a β CACCC box also recruits the same subcomplex as described above. However, the EKLF-bound subcomplex is different from the subcomplex normally interacting with the γ gene transcriptional machinery and fails to assemble with other components, resulting in failure of initiation of γ gene transcription. (C) Putative γ CACCC box-binding factor recruits an appropriate subcomplex of the transcriptional machinery of the γ gene. The appropriate assembly of the initiation complex on the γ gene gives rise to high-level γ gene transcription.