Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease

Abstract

Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease.

Figure 1. Cross species comparative sequence alignment.

(A) Genomic alignment across 11 kb of human and murine DNA containing the Zic3 gene. Known and putative Zic3 exons, untranslated regions (UTR) and non-coding sequences (CNS) are indicated. All exons are conserved at a minimum 91% identity between human and murine sequences. (B) Sequence alignment of the putative exon 4 in human and mouse demonstrates 97% identity.

Figure 2. Genomic structure and alternative splicing of Zic3.

(A) Schematic depiction of the genomic structure of Zic3. Zic3-A consists of exons 1, 2, and 3 while Zic3-B consists of exons 1, 2, and 4. Primer sites used for isoform specific amplification in exons 2–3 and exons 2–4 are indicated. F  =  forward, R(-A)  =  reverse for Zic3-A, and R(-B)  =  reverse for Zic3-B isoforms. (B) Sequence alignment of exon 4 translation across species. (*) identical, (:) functionally conserved or (.) semi-conserved amino acid residues are indicated. (C) Sequence alignment of murine Zic3-A and Zic3-B protein isoforms. Zinc finger domains (ZF-1–5) are indicated by brackets. NLS regions are underlined and NES region is shaded in gray. The last two amino acids in the fifth zinc finger domain that differ between the two isoforms are indicated in red. ZF, zinc finger.

Figure 3. Alignment of the coding sequences of Zic3-A and Zic3-B transcripts.

The coding sequences of murine Zic3-A and Zic3-B were aligned. Alternating exons are indicated by gray shading. The last exons aligned are exons 3 and 4 for Zic3-A and -B, respectively, demonstrating less than 35% identity.

Figure 4. Comparative expression profile of Zic3-A and Zic3–B.

(A–B) Semi-quantitative RT-PCR results of Zic3 isoform specific mRNA expression. (A) Developmental expression profile at E10.5, 13 and 16, and (B) postnatal tissue specific expression profile. (C) Expression of Zic3-A and -B mRNA in HEK293 and P19 cell lines. Approximate band sizes in panels A and C are indicated by arrowheads. Upper  = 300 bp, lower  = 200 bp. cer, cerebellum; mus, skeletal muscle; liv, liver; spl, spleen; brn, brain without cerebellum; hrt, heart; kid, kidney; lng, lung; -RT, control without reverse transcriptase.

Figure 5. Expression of ZIC3-A and ZIC3-B isoforms in mammalian cells.

(A) Anti-ZIC3 immunogen specific for C-terminus (C12), and (B) for N-terminus (N19). Detection of endogenous ZIC3-A and –B expression in (C–D) murine NIH/3T3 and (E–F) human fibroblast whole cell lysate using anti-ZIC3 C-terminus (C, E) or anti-ZIC3 N-terminus specific antibodies (D, F).

Figure 6. Subcellular localization of ZIC3-A and ZIC3-B.

ZIC3 isoform expression in HeLa cells was detected by immunohistochemistry using a mouse anti-FLAG antibody. (A–C) Subcellular localization of ZIC3-A. (D–F) Subcellular localization of ZIC3-B. (G) Quantitative analysis of subcellular localization. Percentages are representative of at least 100 cell counts each from a minimum of six separate experiments.

Figure 7. Transcriptional activation mediated by ZIC3 isoforms.

NIH/3T3 cells were transfected with Flag-GLI3, HA-ZIC3-A, HA-ZIC3-S43X mutant, or HA-Zic3-B. Transfection of ZIC3-A or Zic3-B alone resulted in similar levels of transcriptional activation above baseline, but GLI3 or ZIC3-S43X alone did not result in activation. Co-transfection of ZIC3-A or ZIC3-S43X with GLI3 resulted in co-activation, but there was no increase in activation when Zic3-B and GLI3 were co-transfected. ZIC3-A acts as a co-activator with GLI3 but Zic3-B does not. (*) Significant difference determined by two-tailed, unpaired Student's t-test assuming unequal variances.