Lineage-Specific Expression of Mouse Abcg2 during Hematopoiesis Is Regulated by Alternative Use of Multiple Leader Exons and Promoters.

ABCG2 encodes a transmembrane transporter associated with multi-drug resistance in various cancer cells. ABCG2 is also highly expressed in hematopoietic stem cells (HSCs) and is down-regulated in most committed progenitors, while expression is sharply up-regulated during erythroid differentiation and significant levels of protein expression are detected on mature red blood cells. The expression of ABCG2 in these hematopoietic cells likely reflects a protective role against both endogenous and exogenous toxins. The mechanisms for regulation of ABCG2 expression in hematopoietic cells are poorly understood. We have recently identified three novel leader exons (termed E1a, E1b and E1c) located in the 5′ untranslated region (5′-UTR) of mouse Abcg2 mRNA by EST database searches and reverse-transcription PCR (RT-PCR). In genomic DNA, the distance of these noncoding exons from exon 2, which contains the translation start codon, is 58.5 kb for E1a, 15.0 kb for E1b and 5.1 kb for E1c. In a mouse erythroid cell line (MEL), RT-PCR analysis showed that the transcript containing E1b exon was the only isoform detected. Furthermore, in primary Ter119+ erythroid cells from mouse bone marrow (BM), real time PCR showed that expression levels of the E1b-containing transcript were at least 100 fold greater than those of the E1a transcript. For comparison, cDNA was isolated from c-Kit+, Sca-1+, Lin- (KSL) BM cells, a population devoid of erythroid cells but highly enriched for repopulating HSCs. Real-time PCR showed that the E1a-containing transcript was the major expressed isoform in KSL cells, while the E1b transcript was present at significantly lower levels. The differential expression pattern of Abcg2 mRNA isoforms in HSCs and erythroid cells indicates that at least 2 different promoters control Abcg2 transcription during hematopoiesis. We also analyzed the human EST database and found 3 different ESTs all containing exon 2, similar to mouse Abcg2 gene. RT-PCR analysis of cDNA derived from human placenta and BM mononuclear cells showed expression of all 3 isoforms, which was confirmed by plasmid cloning and sequencing. These results indicate that the human ABCG2 locus also has three leader exons which are alternatively used and give rise to three isoforms of ABCG2 mRNA that differ in their 5′-UTR. We are currently defining the expression pattern of these isoforms in different human hematopoietic cell subpopulations. In summary, our data show the expression of Abcg2 during hematopoiesis is regulated at the level of transcription by alternative use of multiple noncoding leader exons in lineage-specific manner. Functional analysis of different putative promoter regions is underway and should provide a better understanding of the developmental regulation of Abcg2 expression in hematopoiesis.