The strikingly polymorphic DAT phenotype in humans and the biological mechanisms that connect individual genetic backgrounds with the phenotypes still are understood poorly. The functional demands on the DAT1 regulation are seemingly contradictory since the vital necessity of assuring the robust functioning of the dopaminergic system coincides with the need for plasticity and rapid fluctuations in the transcription rate. To better understand the distinct sources and mechanisms that might facilitate a phenotypic diversity of this magnitude, we comprehensively interrogated the sequence of the SLC6A3 gene applying computational methods.
Our analysis revealed several unique features of the human SLC6A3 gene sequence, including (1) a very high frequency of SNPs (897, as in NCBI SNP) compared with the SLC6A4 (441) and other brain-related genes, such as BDNF and DRD4 (454 and 154 SNPs, respectively), that, depending on their positional effect, engender various biological consequences; (2) the abundance of VNTRs (more than 90 in the SLC6A3 gene body alone), which is indicative of a tendency to open chromatin structure in the locus, and increased accessibility to chromatin modifiers; and (3) presence of intragenic CNVs. Most notable characteristic of the human SLC6A3 is its high sensitivity of to epigenetic regulation: in contrast to the relative enrichment in GC nucleotides in the promoter-proximal region as occurs in most human genes, the entire SLC6A3 locus has GC-bias sequence composition (0.55) and comprises multiple CpG sites comprising 27 bona fide CGIs (CpG islands).
Importantly, our analysis revealed that the distinctive sequence features of the SLC6A3
gene recognized by an array of regulatory mechanisms are evolutionally recent
. In fact, they are either uniquely human or shared between human and primates. We noted that the epigenetic sensitivity of the DAT1
gene increased during evolution, and this process likely involves GC-bias gene conversion (GCBC) that is viewed as a major force driving genome evolution 
. Indeed, whilst the mouse Slc6A3
gene has GC content 0.46 and its promoter has no CpG island, promoter-overlapping CpG island is present in the rat's Slc6a3
gene; furthering this tendency, the rhesus monkey's SLC6A3
also has two intragenic CpG islands. During evolution, the increase in demands on the DAT1
expression might well have driven changes in the gene sequence, expanding its sensitivity to the different regulatory mechanisms. In this view, the GC-density and the number of CGIs in the DAT gene
might co-evolve with the growing role of epigenetic mechanisms in its control. In other words, the evolutional drift of the SLC6A3
sequence towards accumulating GC-nucleotides might reflect its enhancing epigenetic potential, needed to accommodate regulatory demands, as dictated by the increasingly complex functions of the human brain.
The genomic regions experiencing GCBC are seen as recombination hotspots 
and, as such, the inherent instability of the SLC6A3
locus could explain its unique sequence features and regulatory attributes. These changes might be linked in time to the genome rearrangement event that resulted in the translocation of a syntenic block encompassing the DAT
gene from the chromosome 13 (rodents) to the telomere-proximal region of the chromosome 5 (human and primates). In general, the propensity of genomic regions to instability is deleterious characteristic, but, theoretically, hypervariability arising from SNPs and tandem repeated sequences could be beneficial by allowing rapid adaptation 
. Accordingly, the high variability of the DAT
locus might support a broad basis for phenotypic diversity, and thus, be advantageous. Our detection of co-occurrence of the SNPs and the repeated sequences with the CGIs (116 SNPs and 2 tandem repeats) infers that the DAT1
might still be evolving.
Our observation that there are several CNVs in the SLC6A3
locus is not unique; they are detected in several human genes 
that seemingly can asymptomatically tolerate this type of genetic variations. Here the human CNV genes stand apart; evidence from animal studies demonstrated that CNVs are depleted from laboratory mouse strains during selective breeding 
. Consequently, it was postulated that that natural selection acts discriminately among human CNV genes and thus, at least for a subset of human genes, CNVs might confer adaptive benefits 
If propensity of the DAT1 locus to instability is high and can give rise to frequent mutations, there must be other means to compensate for potentially sub-optimal gene function resulting from deleterious changes in its sequence. Considering the main concept of systems biology, that emphasizes the biological networks and hubs rather than a single specific process, we posit that the multi-modality of the DAT expressional control with multiple feed-back control loops ensures its functional robustness. Our analysis suggests that the several arms of gene regulatory network modulate the expression of the SLC6A3, whereby differential engagements and/or combinatorial interplay of different mechanisms of this network, DAT expression can be robust and reliable yet plastic and adjustable to accommodate environmental demands.
The novel regulatory paradigm for the SLC6A3
gene that built upon our results might have direct practical implication. Acknowledging that DAT regulation is far more complex than that of average human gene implies that a comprehensive investigation is needed to reliably predict functional changes associated with the DAT
polymorphism(s) and to infer possible phenotypic manifestations of sequence variation(s),. That is, we should consider broadening the scope of the future studies by interrogating the multiple features of the SLC6A3
gene rather than focusing on any single polymorphism in its sequence. Also, the functional- and anatomical- diversity of the brain's dopamine system 
must be accounted for. We elaborate this point taking the 3′-UTR VNTR polymorphism as an example. This polymorphism was investigated in relation to a variety of behavioral traits and diseases 
, and the findings of those research are conflicting. That is, the 10 repeats-allele (10/10 genotype) in some studies, i.e., 
was associated with a lower translation rate and increased extracellular dopamine; while in the others, i.e., 
with higher efficacy of the DAT translation in vitro
and in vivo 
. We suggest that considering a genomic position of the respective polymorphism might resolve this controversy. Indeed, the repeats number (the length of the polymorphic region can affect exclusively the longer mRNAs since the VNTR resides in the middle of 3′-UTR. Evidently, the shorter transcript isoforms (akin to S44625) are insensitive to this variation since here the transcription is terminated upstream of the VNTR. Considering the transcription bias towards preferential production of shorter isoforms in specific brain regions, we expect that a subset of brain functions executed through the specific brain regions is inherently independent of the polymorphism. Conversely, for the behavioral traits that rely on the transcription of long mRNA isoforms, innate differences in transcripts stability and conformational folding, subordinate to the genotype, will be manifest phenotypically.
We want to reiterate that the analyses and observations presented here are theoretical by nature and require experimental validation. However, we believe, that they help broaden our current understanding on the complex regulatory mechanisms imbedded in the human DAT gene sequence.