Identification and functional characterization of an erythroid-specific enhancer in the L-type pyruvate kinase gene.
The rat L-type pyruvate kinase gene is transcribed either from promoter L in the liver or promoter L' in erythroid cells. We have now cloned and functionally characterized an erythroid-specific enhancer, mapped in the fetal liver as hypersensitive site B (HSSB) at 3.7 kilobases upstream from the promoter L'. Protein-DNA interactions were examined in the 200-base pair core of the site by in vivo footprinting experiments. In the fetal liver, footprints were revealed at multiple GATA and CACC/GT motifs, whose association is the hallmark of erythroid-specific regulatory sequences. Functional analysis of the HSSB element in transgenic mice revealed properties of a cell-restricted enhancer. Indeed, this element was able to activate the linked ubiquitous herpes simplex virus thymidine kinase promoter in erythroid tissues. The activation was also observed in a variety of nonerythroid tissues known to synthesize GATA-binding factors. In the context of L'-PK transgenes, HSSB was not needed for an erythroid-specific activation of the L' promoter, while it was required to stimulate the L' promoter activity to a proper level. Finally, HSSB cannot be replaced by strong ubiquitous viral or cellular enhancers, suggesting a preferential interaction of the HSSB region with the L' promoter.
Lacronique
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Institut Cochin de Génétique Moléculaire, Unité 129 INSERM, Université René Descartes, Paris, France.
Lopez
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S
Miquerol
L
L
Porteu
A
A
Kahn
A
A
Raymondjean
M
M
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D005333:Fetus; D015698:Genomic Library; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011770:Pyruvate Kinase / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D014157:Transcription Factors; C085482:Zbtb10 protein, rat; D011770:Pyruvate Kinase; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases