Human zinc finger protein 161, a novel transcriptional activator of the dopamine transporter.
The dopamine transporter (DAT) terminates dopaminergic neurotransmission via reuptake of released dopamine into presynaptic neurons. We have cloned 2.5 kb of the regulatory region upstream of human DAT (hDAT) and constructed a series of deletion mutants to test promoter activity. A comparison of promoter activity between non-neural and neuronal cell lines reveals an interesting difference in pattern. In the PC12 cell line, activity of the proximal promoter is strongly silenced by one or more unidentified elements spanning positions -395 to -2465 of the hDAT gene. Our studies focus on identifying and characterizing the activating factor for hDAT transcription in the sequence between -2511 and -2492 (5(')-CTA CCT GCA CAG TTC ACG GA-3('), termed HY1). In this investigation, we cloned the zinc finger protein 161 (ZFP161) gene as a HY1-binding factor, using the yeast one-hybrid screen. Recombinant ZFP161 was produced to evaluate the DNA-binding properties of the protein. The ability of ZFP161 to directly bind HY1 was examined in an electrophoretic mobility shift assay. RT-PCR analyses revealed that transfection of ZFP161 induced hDAT mRNA expression in HEK293 cells. We additionally confirmed the expression and localization of the DAT protein, using a specific antibody. Both the HY1 sequence and the downstream region were necessary for activation of the hDAT promoter by ZFP161. This finding suggests that the site of cofactor interaction with ZFP161 may exist downstream of HY1.
Lee
Ki-Hwan
KH
Department of Biochemistry, College of Medicine, Hanyang University, Seoul, Republic of Korea
Kwak
Young-Don
YD
Kim
Dong-Hwan
DH
Chang
Mi-Yoon
MY
Lee
Young-Seek
YS
Lee
Yong-Sung
YS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers / Q000235:genetics
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; D015534:Trans-Activators; D014157:Transcription Factors; C088395:VEZF1 protein, human