Zinc Finger and X-Linked Factor (ZFX) Binds to Human SET Transcript 2 Promoter and Transactivates SET Expression

Abstract

SET (SE Translocation) protein carries out multiple functions including those for protein phosphatase 2A (PP2A) inhibition, histone modification, DNA repair, and gene regulation. SET overexpression has been detected in brain neurons of patients suffering Alzheimer's disease, follicle theca cells of Polycystic Ovary Syndrome (PCOS) patients, and ovarian cancer cells, indicating that SET may play a pathological role for these disorders. SET transcript 2, produced by a specific promoter, represents a major transcript variant in different cell types. In this study, we characterized the transcriptional activation of human SET transcript 2 promoter in HeLa cells. Promoter deletion experiments and co-transfection assays indicated that ZFX, the Zinc finger and X-linked transcription factor, was able to transactivate the SET promoter. A proximal promoter region containing four ZFX-binding sites was found to be critical for the ZFX-mediated transactivation. Mutagenesis study indicated that the ZFX-binding site located the closest to the transcription start site accounted for most of the ZFX-mediated transactivity. Manipulation of ZFX levels by overexpression or siRNA knockdown confirmed the significance and specificity of the ZFX-mediated SET promoter activation. Chromatin immunoprecipitation results verified the binding of ZFX to its cognate sites in the SET promoter. These findings have led to identification of ZFX as an upstream factor regulating SET gene expression. More studies are required to define the in vivo significance of this mechanism, and specifically, its implication for several benign and malignant diseases related to SET dysregulation.

Keywords: I2PP2A (protein phosphatase 2A inhibitor); SET (SE Translocation); ZFX (Zinc finger and X-linked transcription factor); gynecologic cancers; transcriptional regulation.

Figure 1

Activities of truncated SET (SE Translocation) transcript 2 promoters in HeLa and HEK 293 cells. (A) Schematic presentation of serial deletions in human SET transcript 2 promoter. Promoters of different lengths (P1 to P8) were subcloned into the pGL3-Basic luciferase vector. TSS: Transcription start site; (B,C) Luciferase activities in transfected HeLa and HEK 293 cells, respectively. Cell transfection was performed with 100 ng of reporter plasmid DNA and 2.5 ng of pRL-TK reference plasmid DNA in 96-well plates. At 24 h post-transfection, firefly and renilla luciferase activities were measured. The firefly luciferase activity was standardized with the correspondent renilla luciferase activity. Data is presented as means ± SD from three independent experiments. (*, p < 0.05).

Figure 2

Identification of crucial transcription factors controlling SET transcript 2 expression. (A) Positions and sequences of cis-elements in the SET core promoter (−157/+47). The transcription start site is indicated by an arrow; (B) HeLa cells were co-transfected with 100 ng of P5 plasmid and 100 ng of plasmid expressing transcription factors. At 24 h post-transfection, cells were lysed and luciferase activities were measured. Data is presented as means ± SD from three independent experiments. (*, p < 0.05).

Figure 3

ZFX transactivates human SET transcript 2 promoter. (A) HeLa cells were transfected with 100 ng of pEF1-ZFX or pEF1-vector (control). At 48 h post-transfection, total RNA and proteins were isolated and ZFX expression was determined. Top panel: Results of real-time PCR showing a dramatic increase of ZFX RNA mRNA following ZFX overexpression. Bottom panel: Results of Western blotting showing an increased ZFX protein expression. GAPDH protein expression was determined and the results indicated equal protein loading; (B) Dose-dependent effect of ZFX on SET promoter activity. HeLa cells were co-transfected with increasing amounts of pEF1-ZFX (0–100 ng, pEF1-vector was used as a “stuffer” to keep a constant DNA amount) and 100 ng of P5 or P7 reporter plasmid. Luciferase activity was measured at 24 h post-transfection. Quantitative data is presented as means ± SD from three independent experiments. (*, p < 0.05).

Figure 4

Mutagenesis analysis of putative ZFX-binding sites in SET transcript 2 promoter. (A) Locations and sequences of the four ZFX putative binding sites. Note the overlapped Site2 and Site3. Mutated sites, designated as M1–M4, were marked with shadows; (B) The left part illustrates the structure of reporter constructs containing mutated sites M1–M4, while the right part shows reporter activities from transfection assays. Reporter activities are expressed as folds of change compared to wild type promoter P5; (C) HeLa cells were co-transfected with the wild type P5 or individual mutant promoter constructs and pEF1-ZFX or the pEF1 control vector. Significantly increased reporter activity was found in P5 and M1-3, but not M4 upon ZFX overexpression; (D) Transient ChIP assay using cell transfected with either wild type P5 or mutant M4 construct. ZFX-specific antibody was applied as described under Materials and Methods. Top panel: A representative image of ChIP results. A clear 322 bp DNA band representing ZFX binding was detected in cells transfected with wild type P5 promoter but not in cells transfected with M4 mutant construct. PCR using DNA templates isolated without immunoprecipitation was used as an input control. PCR using DNA isolated with application of IgG was used as the negative control; Bottom panel: Following densitometry analysis, signals of ZFX binding were standardized by those of input, setting the input value as 1. The quantitative data is presented as means ± SD from three independent experiments. (*, p < 0.05).

Figure 5

Removal of ZFX-binding site led to a loss of response to ZFX knockdown. (A) HeLa cells were transfected with either the non-specific siRNA (Si-Ctl) or one of four ZFX-targeting siRNAs (Si-ZFX1^#, Si-ZFX2^#, Si-ZFX3^# and Si-ZFX4^#). Si-ZFX2^# and Si-ZFX3^# were found to be the most effective for ZFX knockdown. Top panel: Measurement of ZFX mRNA levels. Bottom panel: Western blotting detection of ZFX protein, with GAPDH detected as a control for protein loading; (B) When ZFX expression was knocked down, a decreased reporter activity was observed in the P5 construct containing the Site4 ZFX-binding sequences. No change of reporter activity was observed in P7 in which Site4 was deleted. Similarly, no change in reporter activity was observed in M4 in which Site4 sequences were mutated. Results are presented as means ± SD from three independent experiments. (*, p < 0.01).

Figure 6

Binding of ZFX to the SET transcript 2 promoter confirmed with ChIP assay. ChIP experiments were conducted using ZFX antibody (2 µg) or Flag antibody (2 µg). In (A–C), the top panels show representative results of ChIP. The bottom panels present results of densitometry analysis based on three independent experiments. ZFX-binding results were standardized by those of input, setting the input value as 1. Data is presented as means ± SD from three independent experiments. (*, p < 0.01). (A) When untransfected HeLa cells were used in the ChIP assay, ZFX binding to endogenous SET promoter was readily detected; (B) Upon ZFX knockdown using Si-ZFX2^#, a significant reduction in ZFX binding to endogenous SET promoter was observed; (C) ZFX binding to endogenous SET promoter was detected in cells transfected with ZFX-expressing pEF1-ZFX or control pEF1-vector, and Flag antibody detected a strong binding of SET promoter by overexpressed ZFX.