Abstract 894: Identification of a Novel Mouse GATA5 Transcription Start Site
GATA5 is a zinc finger transcription factor that is expressed in primitive endoderm and precardiac mesoderm during early development, but dramatically decreased in postnatal heart. Previous reports indicate that null mutation of the GATA5 gene in zebrafish results in embyronic lethality, but surprisingly deletion of exon 1 from the mouse GATA5 gene reportedly resulted only in derangement of urogenital development in female mice. This finding suggested the possibility that an alternate GATA5 isoform that lacks exon 1 might appear in mice, and might subserve GATA5 function. To test this possibility, we performed northern analysis and demonstrated that adult mouse lung contains three mRNAs of ~ 4.1, 2.4, and 1.6 kb that hybridize with a probe derived from exons 2 to 6. Interestingly, the smallest mRNA does not hybridize with a probe from the 5′ UTR in exon 1, suggesting that this transcript is generated from an alternate promoter. To identify the transcription start sites of alternative GATA5 transcripts, 5′RLM-RACE was performed using mouse lung mRNA as template, with an RT antisense primer from exon 5 and nested antisense PCR primer in exon 3. Besides recovering the classic start site in exon 1, we discovered a novel transcription start site in intron 1, at 82 bp upstream from exon 2; the resulting mRNA is predicted to encode an N-terminally truncated “short GATA5” comprising aa 225– 404. RT-PCR confirmed that this novel GATA5 transcript also expressed in adult mouse heart. To determine whether both protein isoforms are synthesized, we performed western analysis of whole lung lysate from an E17.5 dpc embryo using a polyclonal antibody directed against aa 235–247. Two immunoreactive bands (~50 kD and ~27 kD) were present, and were of comparable size to those detected in lysates of cells transfected with expression vectors encoding full-length or short GATA5. We also found that bp 889 to 2312 (from GATA5 intron 1 and exon 2) drives luciferase expression during transient transfection in cultured airway myocytes and HEK293 cells. Finally, we found that short GATA5 retains partial ability to transactivate the ANF promoter, which is responsive to full-length GATA5. Together, these data suggest that a partially functional short mouse GATA5 is expressed from an intronic promoter.