Regulation of carcinogenesis through multiple zinc fingers in ZBTB20

Abstract

Zinc finger (ZF) proteins regulate transcription by interacting with cis-acting elements for gene expression in response to stimuli within physiological systems. Bioinformatic studies have proposed that zinc finger and BTB (Broad complex, Tramtrack, Bric-à-brac) domain-containing protein 20 (ZBTB20) acts as a key regulator of crucial genes associated with carcinogenesis. However, biochemical experiments using purified proteins remain unreported. In this study, we investigated the regulatory functions of the ZF domains in ZBTB20, which has five CX₂CX₁₂HX₃H-type classical ZF domains, in the inhibition and expression of downstream transcription factors, including alpha-fetoprotein (AFP) and forkhead box transcription factor O1 (FOXO1). The four ZF domains of ZBTB20, ZBTB20(ZF1-4), inhibit the expression of AFP with specificity (K _d = 0.6 ± 0.04 nM) by interacting with the afp promoter (5'-ACCTA). Furthermore, ZBTB20(ZF1-4) or ZBTB20(ZF1-5) inhibited the expression of FOXO1, thereby suppressing cell cycle arrest and inducing tumorigenesis by binding to the promoter of foxO1 (5'-ACCGCCGCCTC) with affinities of 1.7 ± 0.07 and 2.1 ± 0.05 nM, respectively. The results provide the first evidence that ZBTB20 regulates gene expression through ZF domains positioned at the C-terminus through interactions with cis-acting elements to achieve specificity and selectivity. The balance of ZBTB20 expression can be a crucial issue for the regulation of two downstream transcription factors to maintain homeostasis.

Fig. 1. ZBTB20 from Homo sapiens. (a) Domain organization of ZBTB20. The N-terminal BTB/POZ domain is followed by five classical ZF domains (ZF1–ZF5). The linker region between ZF4 and ZF5 comprises 25 amino acids, which is longer than the typical linkers in classical ZF proteins. (b) Sequence alignment of five classical ZF domains in ZBTB20. Each color denotes an identity (blue), similarity (green), and mismatch (gray) sequence. Yellow highlights indicate cysteine (Cys) and histidine (His) residues involved in zinc ion coordination, characteristic of the CX₂CX₁₂HX₃H-type zinc finger motif.

Fig. 2. ZBTB20-mediated suppression of the alpha-fetoprotein (AFP). (a) Promoter region of Homo sapiens afp gene. The yellow highlighted sequence represents the core ZBTB20 binding site, located before the transcription start site (TSS). (b) and (c) Binding affinities of the wild-type (WT) afp promoter and its mutants with (b) ZBTB20(ZF1-4) and (c) ZBTB20(ZF1-5), measured via fluorescence anisotropy. Error bars represent standard deviations from a minimum of at least three independent experiments.

Fig. 3. ZBTB20-mediated suppression of the forkhead box transcription factor O1 (FOXO1). (a) Putative binding site for ZBTB20 located upstream of foxO1. To identify specific binding sites for the ZBTB20 ZF domains, the foxO1 promoter was divided into F1–F4 segments. The foxO1-F1 region was further divided into S1–S3 segments for a more detailed analysis, as shown below the promoter map. (b) and (c) Binding affinities of the foxO1 promoter sections (F1–F4) with (b) ZBTB20(ZF1-4) and (c) ZBTB20(ZF1-5), measured via fluorescence anisotropy. (d) and (e) Binding affinities of the subdivided foxO1-F1 regions (S1–S3) with (d) ZBTB20(ZF1-4) and (e) ZBTB20(ZF1-5), respectively. The analysis identifies specific binding preferences within the foxO1-F1 region. Error bars indicate standard deviations from a minimum of three independent experiments.

Fig. 4. Transcriptional regulatory interactions of ZBTB20 with cis-acting elements. (a) and (b) Binding curve of cis-acting elements (afp, foxO1-S2, and irs) with (a) ZBTB20(ZF1-4) and (b) ZBTB20(ZF1-5), respectively. Error bars indicate standard deviations from at least three independent experiments.

Fig. 5. Schematic of ZBTB20 zinc finger (ZF) binding to cis-acting elements in cancer regulation. (a) and (b) ZBTB20 ZF domains bound to (a) afp promoter contributing to tumor suppression, and (b) foxO1 promoter facilitating tumorigenesis.