Upstream elements involved in vivo in activation of the brain-specific rat aldolase C gene. Role of binding sites for POU and winged helix proteins.
The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a 115-base pair (bp) promoter fragment was able to ensure the brain-specific expression of the chloramphenicol acetyltransferase (CAT) reporter gene in transgenic mice, but only at a low level (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). Here we show that in vivo activation of this promoter at a high level requires cooperation between an upstream 0.6-kilobase pair (kb) fragment and far upstream sequences. In the 0.6-kb region, a 28-bp DNA element is shown to include overlapping in vitro binding sites for POU domain regulatory proteins and for the Winged Helix hepatocyte nuclear factor-3beta factor. An hepatocyte nuclear factor-3beta-binding site previously described in the short proximal promoter fragment is also shown to interact in vitro with POU proteins, although with a lower affinity than the 28-bp motif. Additional binding sites for POU factors were detected in the upstream 0.6-kb sequences. Progressive deletion in this region resulted in decreased expression levels of the transgenes in mice, suggesting synergistic interactions between these multiple POU-binding sites. We propose that DNA elements characterized by a dual binding specificity for both POU domain and Winged Helix transcription factors could play an essential role in the brain-specific expression of the aldolase C gene and other neuronal genes.
Skala
H
H
Institut Cochin de Génétique Moléculaire, INSERM U129, Université René Descartes, 75014 Paris, France. skala@cochin.inserm.fr
Porteu
A
A
Thomas
M
M
Szajnert
M F
MF
Okazawa
H
H
Kahn
A
A
Phan-Dinh-Tuy
F
F
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D016384:Consensus Sequence; D019175:DNA Methylation; D004789:Enzyme Activation; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D005634:Fructose-Bisphosphate Aldolase