Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.
The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.
Dipartimento di Scienze Cliniche e Biologiche, Ospedale San Luigi Gonzaga, Reg. Gonzole 10 10043, Orbassano Torino, Italy.
A
A
Ceccarelli
N
N
Zhukovskaya
T
T
Kawata
S
S
Bozzaro
J
J
Williams
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D016054:DNA, Protozoan / Q000235:genetics* / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism
D016054:DNA, Protozoan; D016326:Extracellular Matrix Proteins; D015800:Protozoan Proteins; D015534:Trans-Activators; C069214:ecmB protein, Dictyostelium