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| Title |
IDENTIFICATION OF A NEURON-RESTRICTIVE SILENCING
ELEMENT IN THE KCC2 COTRANSPORTER GENE. |
| Author |
M.F. Karadsheh, J. Lu and E. Delpire |
| Affiliation |
Pediatric Critical Care & Anesthesiology
Research Divisions, Vanderbilt University Medical Center, Nashville, Tennessee |
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KCC2 is a neuronal-specific isoform of the K-Cl
cotransporter. It plays a role in GABAergic and glycinergic neurotransmission through its
effect on intracellular Cl- in neurons. To understand the molecular basis for neuronal
specificity of KCC2 expression, we isolated and sequenced portions of the KCC2 gene,
including its 5' flanking (promoter) region. We found a 21 bp sequence within intron 1
that shares 80% homology to the consensus site for NRSF (neuron-restrictive silencing
factor) binding. We demonstrated that this specific sequence of the KCC2 gene promotes
transcriptional regulation, by 1) showing that nuclear proteins isolated from rat brain
interact with this 21 bp element and 2) establishing that this element is able to silence
reporter gene expression in non-neuronal cells. Electrophoretic mobility shift assay was
performed using nuclear protein isolated from rat brain and 32P-labeled double-stranded
NRSE oligonucleotide. We demonstrated the presence of a protein/NRSE DNA complex that
could be displaced with an excess of cold NRSE oligonucleotide but not with unspecific
DNA. This results supports the idea that the short KCC2 sequence interacts with nuclear
proteins or transcription factors. We then analyzed the function of the 21 bp KCC2 element
on transcriptional activity by using a reporter gene. C17 progenitor neural cells were
transfected with 1) a promoterless luciferase vector, 2) a luciferase vector containing
1.5 kb of 5' flanking region of the KCC2 gene, and 3) a vector of same composition but
also containing the 21 bp neuronal-restrictive silencing element found in inton 1. We
showed that the presence of the 1.5 kb fragment upstream of exon 1 significantly increased
luciferase activity when compared to the promoterless vector. Addition of the NRS element
returned the activity of luciferase to baseline values. Taken together, these results
suggest that the 21 bp sequence found in intron 1 of the KCC2 gene is a potential target
for the neuron-restrictive silencer factor and the thus the putative site for KCC2
transcriptional repression in non-neuronal cells. |
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