Visit our Expo - Redox and Inflammation signaling 2012

Session XIV : Transcriptional and translational control Poster XIV, 38
SOX3 expression is driven by multiple CCAAT control elements
Aleksandar Krstic, Milena Stevanovic
Laboratory for human molecular genetics, Institute of Molecular Genetics and Genetic
Engineering, Vojvode Stepe 444a, 11010 Belgrade, Serbia and Montenegro,
E-mail : hmgbox@eunet.yu
Early neurogenesis and neuronal differentiation are precisely controlled by a series of genes.
Sox3 is expressed in the brain from initial stages of development and it is considered to be
one of the earliest markers in vertebrates playing the role in specifying neuronal fate. In order
to elucidate molecular mechanisms underlying the regulation of the human SOX3 gene
expression, computer prediction software was used to search the matrix database and to
identify potential transcription binding sites within the human SOX3 promoter. Among the
number of putative consensus binding sites three evolutionary conserved CCAAT boxes,
representing the putative binding sites for the general transcription factor NF-Y, were
identified at positions –101 bp to –105 bp, -246 bp to -250 bp and -326 bp to -330 bp. EMSA
and “supershift” experiments, for fragments containing each of the identified CCAAT boxes,
were performed to prove the specificity of the NF-Y binding. To examine whether the
putative CCAAT boxes are functional, site directed mutagenesis was performed. The ability
of the single, double and triple site mutants and its wild-type counterpart to drive expression
of the cat reporter gene was examined in stem and RA induced NT2/D1 cells. We have
shown that mutagenesis of all three CCAAT boxes affects the expression of the reporter gene,
compared to the wild-type counterpart. Results obtained with double NF-Y site mutants
suggest functional/spatial interaction of these regulatory elements. Triple NF-Y site mutant
reduced reporter construct activity by almost 20 fold. All results strongly suggest -that all
three CCAAT box motifs present in the SOX3 promoter play functional role as transcriptional
activators of this gene.
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