The ZFHX1A gene is differentially autoregulated by its isoforms

Abstract

The Zfhx1a gene expresses two different isoforms; the full length Zfhx1a-1 and a truncated isoform termed Zfhx1a-2 lacking the first exon. Deletion analysis of the Zfhx1a-1 promoter localized cell-specific repressors, and a proximal G-string that is critically required for transactivation. Transfection of Zfhx1a-1 cDNA, but not Zfhx1a-2, downregulates Zfhx1a-1 promoter activity. Mutation of an E2-box disrupted the binding of both Zfhx1a isoforms. Consistent with this, transfected Zfhx1a-1 does not regulate the transcriptional activity of the E-box mutated Zfhx1a-1 promoter. Competitive EMSAs and transfection assays show that Zfhx1a-2 can function as a dominant negative isoform since it is able to compete and displace Zfhx1a-1 from its binding site and overcome Zfhx1a-1 induced repression of the Zfhx1a-1 promoter in cells. Hence, the Zfhx1a-1 gene is autoregulated in part by negative feedback on its own promoter which is, in turn, modified by the availability of the negative dominant isoform Zfhx1a-2.

Figure 1. Zfhx1a-1 promoter activity in Jurkat, CHO-K1 and C2C12 cells and localization of an essential Zfhx1a-1 promoter enhancer to the G-string

(A) Schematic of DNA constructs containing the 5′-flanking region of Zfhx1a-1 gene in the pGL3 vector. Z1p.786ΔEag1 contains from −913 to −129 of the Zfhx1a promoter inserted into pGL3-promoter vector having the SV40 promoter (stripped box). The bent arrow is the transcriptional start site. (B) Luciferase activity of 5′-deletions of Zfhx1a-1 promoter in Jurkat (immature lymphoblast), CHO-K1 (ovary) and C2C12 (myoblast) cells. Clones were transfected into triplicate wells and the results were expressed as luciferase/β-galactosidase activity. Values are the fold activation compared to Z1p.12Luc, expressed as the mean±SEM of three to six experiments. Means were compared by one-way ANOVA followed by Student-Newman-Keuls. *P<0.05 vs. Z1p.12Luc; **P<0.01 vs. Z1p.12Luc, ***P<0.001 vs. Z1p.12Luc. (C) Deletions of the proximal promoter were transfected into Jurkat cells. Values are the fold activation compared to the appropriate controls (either Z1p.12Luc, or pGL3-promoter), expressed as the mean±SEM of three experiments. ****P<0.0001 vs. Z1p.133Luc. (D) The sequence of the human Zfhx1a promoter analyzed by TFSEARCH (at http://www.cbrc.jp/research/db/TFSEARCH.html) identifies candidate regulatory transcription factors; Runx (R), MZF1 (M), and SP1 (SP) binding sites. The underlined sequence is deleted in the Z1p.133ΔG construct.

Figure 2. Zfhx1a-1 represses its own promoter activity, but Zfhx1a-2 does not

(A) Co-transfection of Zfhx1a promoter clones with Zfhx1a-1 or -2 expression plasmids. Cells were co-transfected with the indicated reporter constructs and Zfhx1a-1 or Zfhx1a-2 expression vectors, or the empty expression vector (pcDNA4/HisMax). The promoter activity of each construct was normalized to the condition with the highest activity. (B) CHO-K1 cells were co-transfected with 1.0 μg of Z1p1000Luc and with 0.5 μg, 0.8 μg, 1.0 μg and 1.3 μg of the Zfhx1a-1 expression vector, or with 1.0 μg of the empty vector. Results are mean±SEM in both figures (n=4–6).

Figure 3. BS2 sequence is necessary for each Zfhx1a isoform binding to Zfhx1a-1 promoter

(A) Zfhx1a is strongly expressed in Jurkat and mouse thymocytes. Western analysis of Zfhx1a protein expression in thymocyte nuclear extract (Thy), and cytosol (Thy(c)) and nuclear extracts from C33a (C33), SupT1 (T1), and Jurkat (J) cells. (B) EMSAs were performed with Jurkat nuclear extracts (NE), Zfhx1a-2 programmed reticulocyte lysate (Zfhx1a-2 RRL) or unprogrammed reticulocyte lysate (UP RRL) plus [³²P]BS 1+2 oligonucleotide. (C) EMSA using the same protein sources and the [³²P]BS1 probe, showing no binding of protein. (D) EMSA using the [³²P]BS2 probe. ³²P-probes were competed by 100-fold molar excess of cold specific oligonucleotides (Unlabeled probe) or cold actin oligonucleotide (Actin oligo). Antibodies used for supershift are a) SC-E20 Zfhx1a N-terminus antibody (E20), b) SC-C20 Zfhx1a C-terminus antibody (C20), and c) anti-Actin antibody (C11). (E) EMSAs with Jurkat NE or Zfhx1a-2 RRL were incubated with the wild type [³²P]BS2 probe (WT ³²P-PROBE) or its mutant (Mut ³²P-PROBE). (F) CHO-K1 cells were co-transfected with 0.8 μg of the Zfhx1a-1, Zfhx1a-2 expression vector or the empty vector and 1.0 μg of the Z1p1000Luc or Z1p1000mut Luc. Results are expressed as mean±SD and are representative of 3 experiments with same results. ***P<0.001 vs. control (first column on the left).

Figure 4. Zfhx1a-2 blocks the silencer activity of Zfhx1a-1 and displaces Zfhx1a-1 from its binding site

(A) EMSA where the [³²P]BS2 probe was incubated with 1.0 μl of Jurkat NE plus increasing amounts of Zfhx1a-2 RRL (1–9 μl). (B) CHO-K1 cells were co-transfected with 0.8 μg of Z1p359Luc, 0.8 μg of the Zfhx1a-1 expression vector and increasing amounts (0.25 μg, 0.5 μg and 0.8 μg) of the Zfhx1a-2 expression vector. Total DNA concentrations were equalized using the empty expression vector. Results are expressed as mean±SD and they are representative of 3 experiments with same results. *P<0.05, ***P<0.001 vs. control (first column on the left).