Controlling gene expression through five zinc finger domains of ZNF18

Abstract

Zinc finger (ZF) proteins are the most abundant transcription factors in vertebrates, and they regulate gene expression through interactions with cis-acting elements. ZF domains selectively recognize specific sequences to accelerate or repress target genes. Zinc finger protein 18 (ZNF18) contains five CX₂CX₁₂HX₃H-type ZFs at the C-terminus, which are expressed in the brain and other organs of the biological system. Bioinformatic study proposed that cyclin-dependent kinase 1 (CDK1) is in the signaling cascade of ZNF18; although experimental evidence has not yet been reported. In this study, we expressed and purified ZNF18(ZF1-5), five ZF domains from ZNF18, and investigated metal binding specificity and promoter interactions. ZNF18(ZF1-5) has specific coordination to Zn²⁺ (K_d ≤ 18 nM) compared with other xenobiotic metal ions, including Co²⁺, Fe²⁺, and Fe³⁺, with 98.5% of reduced ZF domains after purification. This significantly active ZF can be one of the major reasons for tight coordination affinity. CDK1 rescued the arrested cell cycle induced by DNA damage, resulting in tumorigenesis. Zn²⁺-ZNF18(ZF1-5) specifically binds to cis-acting elements of cdk1 (K_d = 4.63 ± 0.07 nM), mediated by a cell cycle-dependent element (cde, 5'-CGCGG) and a cell cycle gene homology region (chr, 5'-TTGAA). The ZNF18 superfamily was expressed in the brain for the regulation of neuronal development and cell differentiation. Zn²⁺-ZNF18(ZF1-5) interacted with promoters in the insulin response sequence (IRS) for inhibition of dopamine secretion and cis-acting element of brain-2 (BRN2), which controlled astrocyte and cancer development. These results provide the first evidence that ZNF18(ZF1-5) regulates the cell cycle and neuronal development through transcriptional regulation.

Keywords: apoptosis; astrocyte; cell cycle regulation; classic zinc fingers; cyclin‐dependent kinase 1; transcription factor; zinc finger protein 18 (ZNF18).

FIGURE 1

ZNF18 from Homo sapiens. (a) Domain organization of ZNF18. (b) Sequence alignment of the five zinc finger domains of ZNF18. Identical and similar amino acid sequences are shown in blue and green, respectively. Bold letters highlighted in yellow indicate sequences related to the Zn²⁺ coordination site.

FIGURE 2

Dynamics of metal‐coordination in ZNF18(ZF1‐5). (a) UV–Vis spectra showing Co²⁺ titration to apo‐ZNF18(ZF1‐5). (b) Increase in the d–d transition band at 643 nm as Co²⁺ coordinates to apo‐ZNF18(ZF1‐5). (c) Determination of the K _d value by curve fitting with the changes in absorption at 643 nm observed during Co²⁺ titration. (d) Non‐specific d–d transition bands at 695 and 743 nm during Co²⁺ titration. (e) Proposed model of Co²⁺ coordination in ZNF18(ZF1‐5) with Cys₄ mis‐coordination at low Co²⁺ equivalents and proper Cys₂His₂ coordination with excess Co²⁺. Experiments were performed with 10 μM ZNF18(ZF1‐5) in 100 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP, N₂ (g)‐filled glovebox, and 25°C.

FIGURE 3

Binding of Zn²⁺ to ZNF18(ZF1‐5) analyzed through competitive binding. (a) UV–Vis spectra showing Zn²⁺ back‐titration to Co²⁺–ZNF18(ZF1‐5). (b) Decrease in the d–d transition band at 643 nm as Co²⁺ is replaced by Zn²⁺ in ZNF18(ZF1‐5). (c) Determination of the K _d value by curve fitting with the changes in absorption at 643 nm observed during the Zn²⁺ back‐titration. Experiments were performed with 10 μM ZNF18(ZF1‐5) in 100 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP, N₂ (g)‐filled glovebox, and 25°C.

FIGURE 4

Oxidation state‐dependent interaction of iron with apo‐ZNF18(ZF1‐5). (a) UV–Vis spectra showing the coordination of Fe²⁺ to apo‐ZNF18(ZF1‐5). (b) Determination of the K _d value by the curve fitting of the absorbance at 309 nm during Fe²⁺ titration to apo‐ZNF18(ZF1‐5). (c) UV–Vis spectra showing Fe³⁺ titration to apo‐ZNF18(ZF1‐5). Experiments were performed with 10 μM ZNF18(ZF1‐5) in 100 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP, N₂ (g)‐filled glovebox, and 25°C.

FIGURE 5

Interaction between ZNF18(ZF1‐5) and the cdk1 promoter. (a) Schematic diagram of CDK1 inhibition by ZNF18 through binding to the CDE and CHR elements. (b) Binding of ZNF18(ZF1‐5) to the cdk1 promoter and its mutated variants. Native and mutated sequences are highlighted in bold black and red, respectively. The K _d values, measured through curve fitting, are presented in the inlet table as averages with standard deviations. Experiments were performed with 5 nM DNA in 25 mM MOPS (pH 7.4), 50 mM NaCl, 1 mM DTT, and 25°C.

FIGURE 6

Binding of ZNF18(ZF1‐5) to cis‐acting elements and its mutated variants. (a, b) Interaction of ZNF18(ZF1‐5) with (a) CDE and (b) CHR. Native and mutated sequences are highlighted in bold black and red, respectively. The K _d values, measured through curve fitting, are presented in the inlet table as averages with standard deviations. Experiments were performed with 5 nM DNA in 25 mM MOPS (pH 7.4), 50 mM NaCl, 1 mM DTT, and 25°C.

FIGURE 7

Regulation of dopamine release and neuronal development by ZNF18(ZF1‐5). (a) Interaction of ZNF18(ZF1‐5) with irs and its mutated variants. (b) Interaction of ZNF18(ZF1‐5) with putative nucleotides to determine the promoter element through fluorescence anisotropy. Putative promoter regions of brn2 are classified (F1–F3) to investigate the interaction between ZNF18(ZF1‐5) and promoter sequences. Experiments were performed with 5 nM DNA in 25 mM MOPS (pH 7.4), 50 mM NaCl, 1 mM DTT, and 25°C.

FIGURE 8

Predicted structure of Homo sapiens ZNF18(ZF1‐5) by AlphaFold2. The molecular surface is colored according to the charge distribution: Red and blue indicate negative and positive charges, respectively.

FIGURE 9

Physiological function of ZNF18 in regulating the cell cycle, tumor progression, and neurodegeneration.