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C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity.

Zhang B, Xiang S, Yin Y, Gu L, Deng D - BMC Biotechnol. (2013)

Bottom Line: Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity.In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China. dengdajun@bjmu.edu.cn.

ABSTRACT

Background: It is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1's DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions.

Results: In the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.

Conclusions: Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

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Results of EMSA assay for the binding of the purified 7ZFPs to the p16-specific Probe-1. (a) The binding activity of 7ZFPs to the Probe-1 DNA was abolished after the S to R mutation in finger 6 in the 7ZFPs Active-G and Active-I; Variant-(f6), variants with a S to R mutation of the key amino acid at the position +2 of the α-helix in finger-6 in 7ZFPs; (b) The binding activity of 7ZFPs to the Probe-1 DNA was abolished when their C-terminal 2ZF domain in was replaced with the middle 2ZF domain in the 7ZFP Active-B and Active-C, or the linker between finger-7 and the 6Histidine-tag in the C-terminal region was shorten from TGEKP to TG in the 7ZFP Active-K.
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Figure 4: Results of EMSA assay for the binding of the purified 7ZFPs to the p16-specific Probe-1. (a) The binding activity of 7ZFPs to the Probe-1 DNA was abolished after the S to R mutation in finger 6 in the 7ZFPs Active-G and Active-I; Variant-(f6), variants with a S to R mutation of the key amino acid at the position +2 of the α-helix in finger-6 in 7ZFPs; (b) The binding activity of 7ZFPs to the Probe-1 DNA was abolished when their C-terminal 2ZF domain in was replaced with the middle 2ZF domain in the 7ZFP Active-B and Active-C, or the linker between finger-7 and the 6Histidine-tag in the C-terminal region was shorten from TGEKP to TG in the 7ZFP Active-K.

Mentions: As previously reported [22], results in the EMSA assays using the purified 7ZFPs showed that the representative 7ZFPs including Active-A to -K had high affinity binding to the p16 promoter-specific Probe-1 (Figures 4, 5, and Table 1). First, we investigated whether substitution of single amino acid mutation (S > R) at position +2 of finger-6′s α-helix in the C-terminal 2ZF could affect the Probe-1 binding affinity. As shown in Figure 4a, compared with Active-G and Active-I, this substitution completely abolished their binding ability with Probe-1 (compare lane 2 and 3, lane 4 and 5, also see Table 1, line 12 to 13 and line 16 to 17). The EMSA experiments with 7ZFPs Active-D, F, H, J, K and their variants also showed the same results (EMSA image not shown, and Table 1, line 8 to 11, line 14 to 15, and line 18 to 21). Second, for 7ZFP Active-K, a deletion (TGEKP > TG) of 3 amino acid residues in the linker between C-terminal finger-7 and the 6 × Histidine-tag also fully destroyed p16 binding (Figure 4b, compare lane 6 and 7, Table 1, line 22 and 23). Next, full depletion of p16 binding activity was also observed for the representative 7ZFPs Active-B and Active-C after their finger-6 and −7 were replaced with finger-4 and −5 (Figure 4b, compare lane 2 and 3, lane 4 and 5; Table 1, compare line 3 and 5, line 6 and 7). In contrast, the replacement of finger-3 at the N-terminal 3ZF domain with finger-5 in 7ZFPs Active-A and Active-B did not affect their p16 binding affinity (Figure 5a, compare lane 2 and 6; Figure 5b, compare lane 2 and 6; Table 1, compare line 1 and 2, line 3 and 4). These results suggest that the C-terminal fingers and flanking sequence may play a crucial role in determining the 7ZFPs’ DNA binding affinity.


C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity.

Zhang B, Xiang S, Yin Y, Gu L, Deng D - BMC Biotechnol. (2013)

Results of EMSA assay for the binding of the purified 7ZFPs to the p16-specific Probe-1. (a) The binding activity of 7ZFPs to the Probe-1 DNA was abolished after the S to R mutation in finger 6 in the 7ZFPs Active-G and Active-I; Variant-(f6), variants with a S to R mutation of the key amino acid at the position +2 of the α-helix in finger-6 in 7ZFPs; (b) The binding activity of 7ZFPs to the Probe-1 DNA was abolished when their C-terminal 2ZF domain in was replaced with the middle 2ZF domain in the 7ZFP Active-B and Active-C, or the linker between finger-7 and the 6Histidine-tag in the C-terminal region was shorten from TGEKP to TG in the 7ZFP Active-K.
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Figure 4: Results of EMSA assay for the binding of the purified 7ZFPs to the p16-specific Probe-1. (a) The binding activity of 7ZFPs to the Probe-1 DNA was abolished after the S to R mutation in finger 6 in the 7ZFPs Active-G and Active-I; Variant-(f6), variants with a S to R mutation of the key amino acid at the position +2 of the α-helix in finger-6 in 7ZFPs; (b) The binding activity of 7ZFPs to the Probe-1 DNA was abolished when their C-terminal 2ZF domain in was replaced with the middle 2ZF domain in the 7ZFP Active-B and Active-C, or the linker between finger-7 and the 6Histidine-tag in the C-terminal region was shorten from TGEKP to TG in the 7ZFP Active-K.
Mentions: As previously reported [22], results in the EMSA assays using the purified 7ZFPs showed that the representative 7ZFPs including Active-A to -K had high affinity binding to the p16 promoter-specific Probe-1 (Figures 4, 5, and Table 1). First, we investigated whether substitution of single amino acid mutation (S > R) at position +2 of finger-6′s α-helix in the C-terminal 2ZF could affect the Probe-1 binding affinity. As shown in Figure 4a, compared with Active-G and Active-I, this substitution completely abolished their binding ability with Probe-1 (compare lane 2 and 3, lane 4 and 5, also see Table 1, line 12 to 13 and line 16 to 17). The EMSA experiments with 7ZFPs Active-D, F, H, J, K and their variants also showed the same results (EMSA image not shown, and Table 1, line 8 to 11, line 14 to 15, and line 18 to 21). Second, for 7ZFP Active-K, a deletion (TGEKP > TG) of 3 amino acid residues in the linker between C-terminal finger-7 and the 6 × Histidine-tag also fully destroyed p16 binding (Figure 4b, compare lane 6 and 7, Table 1, line 22 and 23). Next, full depletion of p16 binding activity was also observed for the representative 7ZFPs Active-B and Active-C after their finger-6 and −7 were replaced with finger-4 and −5 (Figure 4b, compare lane 2 and 3, lane 4 and 5; Table 1, compare line 3 and 5, line 6 and 7). In contrast, the replacement of finger-3 at the N-terminal 3ZF domain with finger-5 in 7ZFPs Active-A and Active-B did not affect their p16 binding affinity (Figure 5a, compare lane 2 and 6; Figure 5b, compare lane 2 and 6; Table 1, compare line 1 and 2, line 3 and 4). These results suggest that the C-terminal fingers and flanking sequence may play a crucial role in determining the 7ZFPs’ DNA binding affinity.

Bottom Line: Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity.In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China. dengdajun@bjmu.edu.cn.

ABSTRACT

Background: It is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1's DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions.

Results: In the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.

Conclusions: Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

Show MeSH