Regulation of human pyruvate kinase gene in red cells

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To identify regulatory gene sequence(s) responsible for the erythroid-specific expression of human erythroid-type pyruvate kinase (R-PK), we prepared two human PK mini-gene constructs and established 19 lines of transgenic mice.Although we have shown that the human R-PK promoter containing 4 GATA and 2 CACCC motifs had prominent transcription activity in both K562 and MEL cells, the promoter was not sufficient for in vivo transcription in the PK transgenic mice.We identified two DNaseI hypersensitive sites (HS) upstream of the human PK LR-gene, and an addition of the proximal HS (HS-I) enabled the linked PK transgene to express in an erythroid-specific manner, suggesting that the HS-I is an erythroid-specific enhancer of the human PK LR-gene.The HS-I contained one GATA, one CACCC and one purine rich motif (AGGGAGAAG), and the organization was similar to the erythroid-specific enhancer which had been identified in rat PK LR-gene.We have established three PK transgenic mice lines which overexpress human R-PK in red cells.The mu' LCR, the regulatory sequence of human beta-like globin locus was linked to the R-PK promoter and coding sequences, and used for microinjection.Red cell PK activities of the PK transgenic mice were about 2-3 fold of normal controls (83-122 IU/gHb, normal range 41.6*5.8), and human R-PK activities were demonstrated in the PK zymograms.Since the mu' LCR did not confer the position-independent, copy number-dependent expression of the PK transgene, the LCR might be influenced by position effect, and functioned as an erythroid-specific enhancer.