Identification of an intronic cis-acting element in the human dopamine transporter gene

Abstract

The human dopamine transporter gene (hDAT) encodes the dopamine transporter in dopamine (DA) neurons to regulate DA transmission. hDAT expression varies significantly from neuron to neuron, and from individual to individual so that dysregulation of hDAT is related to many neuropsychiatric disorders. It is critical to identify hDAT-specific cis-acting elements that regulate the hDAT expression. Previous studies showed that hDAT Intron 1 displayed inhibitory activity for reporter gene expression. Here we report that the hDAT Intron 1 contains a 121-bp fragment that down-regulated both SV40 and hDAT promoter activities by 80% in vitro. Subfragments of 121-bp still down-regulated the SV40 promoter but not the hDAT promoter, as supported by nuclear protein-binding activities. Collectively, 121-bp is a silencer in vitro that might coordinate with transcriptional activities both inside and outside 121-bp in regulation of hDAT.

Identification of HIVEP2 as a dopaminergic transcription factor related to substance use disorders in rats and humans

“…In the human neuroblastoma SK-N-AS cells which served as another model for DA neurons 22 , overexpression of pcDNA3.1 + HIVEP2 increased the HIVEP2 protein levels as determined by Western blotting (Fig. 3a), verifying both the cDNA clone and the anti-HIVEP2 sera.…”

“…Previous study showed that the 121-bp of haplotype A, especially a 36 bp subfragment containing the HIVEP2 target “1”, displayed inhibitory activity only on the SV40 promoter 22 . It was an expectation that we would be able to clone a trans -repressor, instead of a trans -activator (HIVEP2).…”

“…The 121-bp fragment of haplotype A 22 was used as the search target and a human adult brain cDNA library was constructed into pP6, a pGADGH derivative plasmid, both used in Y1H in the search for proteins targeting the 121-bp (Hybrigenics Services, Paris, France). The bait construct was first checked by re-sequencing and then transformed into the yeast strain Y1H300 (MAT a, Gal4Δ, ade2-101, trp1-901, leu2-3,112, his3Δ200), integrating the DNA bait into the yeast genome.…”

hVMAT2: A Target of Individualized Medication for Parkinson's Disease

“…An electrophoretic mobility shifting assay was carried by using nuclear proteins extracted from SH-SY5Y cells, according to our previously published protocol [18,30]. Oligonucleotides used for annealing are listed in Table 1.…”

AZI23’UTR Is a New SLC6A3 Downregulator Associated with an Epistatic Protection Against Substance Use Disorders

“…Luciferase (Luc) reporters SV40-A (pGL3-SV40-A-luc) and SV40-B (pGL3-SV40-B-luc) were constructed by directionally inserting 40 bp dsDNA oligo containing DNPi allele A or B in the middle of the oligo, after self-annealing the oligos carrying two restriction site ends, into Hind III/ Nco I sites of Promega’s pGL3 SV40 Promoter vector (Madison, WI, USA; see Supplementary Table 1 for a list of all nucleotides used in this study). Preparation of 7.9kb constructs has been described before[25]. 7.9kb-A (pGL3-hDAT7.9kb-A) and 7.9kb-B (pGL3-hDAT7.9kb-B) differed only by DNPi alleles and both covered a SLC6A3 promoter region (7.9kb) from 5,931bp upstream of transcription start site to 35 bp downstream of DNPi.…”