Abstract
Uroporphyrinogen III synthase (URO-synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-synthase, the cDNA and approximately 24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5'-untranslated regions (5'-UTRs), but identical coding exons 2B through 10. Transcripts with 5'-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5'-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene.
Highlights
Uroporphyrinogen III synthase (URO-synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria
5Ј-RACE of Mouse Splenic cDNA Demonstrates Multiple URO-synthase 5Ј-UTRs—To assess the possible existence of separate housekeeping and erythroid-specific transcripts for the mouse URO-synthase, 5Ј-RACE was performed using Marathon-Ready cDNA made from mouse splenic mRNA (Fig. 1A)
Of the over 200 5ЈRACE clones identified, there were almost 30 times more group 2A than group 1A clones. Both group 1A and 2A clones had 5Ј-UTRs that differed from 5Ј-UTR of the previously reported mouse URO-synthase cDNA [22], while the 3Ј sequence adjacent to the different 5Ј-UTRs was common in all three groups
Summary
AN ALTERNATIVE PROMOTER CONTROLS ERYTHROID-SPECIFIC EXPRESSION IN THE MURINE GENE*. (Received for publication, April 2, 1999, and in revised form, November 8, 1999). To investigate the erythroidspecific expression of murine URO-synthase, the cDNA and ϳ24-kilobase genomic sequences were isolated and characterized. Housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene. The genomic sequences encoding the eight mammalian (human and/or murine) heme biosynthetic enzymes have been isolated and characterized [1,2,3,4,5,6,7], with the notable exception of URO-synthase, the fourth enzyme in the pathway We report that the mouse URO-synthase gene has distinct erythroid and housekeeping promoters that generate three different transcripts, one being erythroid-specific. The genomic organization, promoter sequences, and 5Ј transcription start sites of the alternative transcripts were determined, and erythroid-specific expression was demonstrated in MEL and human K562 cells
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