Intragenic Transcriptional cis-Antagonism Across SLC6A3

Abstract

A promoter can be regulated by various cis-acting elements so that delineation of the regulatory modes among them may help understand developmental, environmental and genetic mechanisms in gene activity. Here we report that the human dopamine transporter gene SLC6A3 carries a 5' distal 5 kb super enhancer (5KSE) which upregulated the promoter by 5-fold. Interestingly, 5KSE is able to prevent 3' downstream variable number tandem repeats (VNTRs) from silencing the promotor. This new enhancer consists of a 5' VNTR and three repetitive sub-elements those are conserved in primates. Two of 5KSE's sub-elements, E-9.7 and E-8.7, upregulate the promoter but only the later could continue doing so in the presence of VNTRs. Finally, E-8.7 is activated by novel dopaminergic transcription factors including SRP54 and Nfe2l1. Together, these results reveal a multimodal regulatory mechanism in SLC6A3.

Presence of recombination hotspots throughout SLC6A3

“…We now report indeed that Tajima’s D results are consistent with the functionality of DNPi because the EA DNPi was positively selected and this DNPi indeed mediates a long non-coding RNA (lncRNA) regulation of the SLC6A3 promoter[75]. Significantly, SSLP in Intron 1, 5’VNTR and -10kb-pA were all selected in both populations, consistent with the fact that SLC6A3 cis -acting elements such as the 5 kb super enhancer (5KSE) can be localized to distal 5’ regions[95] and that 5’VNTR was positively correlated with the mRNA levels in postmortem dopamine neurons[76]. These positive selections thus generate testable hypotheses for functionality and associations.…”

“…It is important to uncover novel, potentially functional polymorphisms and distinct haplotypes, and recombination hotspots because SLC6A3 activity can be haplotype-dependent partly due to cis -antagonism between 5’ and 3’ sides of SLC6A3 [95]. The most significant findings from this deep-sequencing study included discovery of novel and selected polymorphisms and the presence of recombination hotspots throughout SLC6A3 .…”

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

“…Screening was performed against the random-primed cDNA library. Clones were screened and complete cDNAs were cloned as described before 12 .…”

“…Rabbit anti-HIVEP2 sera were kindly provided by Shunsuke Ishii of Institute of Physical and Chemical Research (RIKEN) in Japan. Methods were as described before 12 . For SK-N-AS cell staining, coverslips were blocked in 7% non-fat milk at RT for 1 h. Coverslips were incubated with rabbit ant-HIVEP2 sera (1:50 diluted in 0.01% BSA in Tris buffered saline (TBS)) or with diluted normal rabbit serum (Gibson Bioscience) as control at 4 ˚C for 48 h. The secondary antibody is Alexa Fluor 568 goat anti-rabbit IgG (1:500 diluted, RRID AB_143157, Invitrogen).…”

Dopamine Genetics: Substance Use Disorders vs. Parkinson’s Disease

“…Determine whether these neuropsychiatric disorders have common risks in DA-associated genes, including DA synthesis (2 genes), metabolism (2 genes), receptors (DRs, 5 genes), monoamine transporters (5 genes), an uptake modulator (alpha-synuclein: SNCA), and transcription factors (7 genes) [11,12].…”