Genetically engineered dopamine beta-hydroxylase gene promoters with better PHOX2-binding sites drive significantly enhanced transgene expression in a noradrenergic cell-specific manner.
A continuously growing body of evidence suggests that dysregulation of noradrenergic (NA) neurons is implicated in the etiology and pathophysiology of various human diseases such as depression, drug addiction, and autonomic dysfunction. An efficient NA neuron-specific promoter is potentially valuable to investigate the precise role of NA neurons in normal as well as in diseased brain and to treat the associated disorders by gene therapy. In this study, we tested a novel strategy to modify genetically the promoter of the human dopamine beta-hydroxylase (hDBH) gene to overcome its inherent weakness while maintaining its cell-type specificity. We optimized the nucleotide sequence motifs of PHOX2-binding sites (PRS2 and PRS3) residing within the hDBH promoter. Optimization of both PRS2 and PRS3 motifs significantly increased their binding affinities to PHOX2A, leading to a dramatic increase in the promoter strength (>20-fold). More importantly, these modifications do not alter the level of transgene expression in non-NA cells either in vitro or in vivo, demonstrating tight cell-type specificity. This work shows that a cellular gene promoter can be genetically modified to strengthen its promoter activity without losing cell-type specificity by optimizing critical cis-regulatory elements. Our genetically engineered promoter may be useful for cell-type-specific gene targeting as well as for generating in vivo animal models with altered gene expression in a specific cell type.
Hwang
Dong-Youn
DY
Molecular Neurobiology Laboratory, McLean Hospital, and Program in Neuroscience, Harvard Medical School, Belmont, MA 02478, USA.
Hwang
Michelle M
MM
Kim
Han-Soo
HS
Kim
Kwang-Soo
KS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D004299:Dopamine beta-Hydroxylase / Q000235:genetics*; D017930:Genes, Reporter; D005818:Genetic Engineering; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics*
D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; C491061:PHOX2A protein, human; D014157:Transcription Factors; D004299:Dopamine beta-Hydroxylase