Screening the optimal activity region of the dopachrome tautomerase gene promoter in sheep skin melanocytes

ABSTRACT To determine the optimal activity region of the dopachrome tautomerase gene (DCT) promoter in sheep skin melanocytes, five eukaryotic expression vectors were constructed containing DCT promoter fragments of different lengths (441 bp, 652 bp, 834 bp, 1155 bp, and 1504 bp) ligated to the pLV.ExSi.P/Puro-mouse DCT-eGFP vector. These constructs were transiently transfected into sheep skin melanocytes using liposome transfection technology. GFP (Green Fluorescent Protein) expression varied among the groups transfected with different constructs as follows: 652 bp > 834 bp > 441 bp > 1504 bp > 1155 bp. GFP expression was significantly higher in the 652 bp and 834 bp groups than in the 1155 bp group (p < 0.01), while expression in the 652 bp group was significantly higher than in the 1504 bp group (p < 0.05). Additionally, we identified elements participating in the initiation of gene transcription (a CAAT box), cell type-specific expression (M-box), and the regulation of gene expression (E-box) in the sheep DCT promoter. In conclusion, the optimal active region of the sheep DCT promoter is located between –593 bp and + 41 bp.


Introduction
Melanin is produced by melanosomes within melanocytes, and is responsible for the mammalian coat colour (Zhang et al. 2017). Several enzymes are involved in catalyzing the synthesis of melanin, such as tyrosinase (TYR), dopachrome tautomerase (DCT or TYRP2), and tyrosinase-related protein 1 (TYRP1) (Woods and Bishop 2011). The absence of tyrosinase leads to an albino phenotype, while mutations of DCT and TYRP1 alter the type of pigment produced . For example, the absence of DCT leads to a dark grey coat colour (Guyonneau et al. 2004). DCT is first expressed in the embryonic retinal pigmented epithelium, then the telecephalon and melanoblasts (Woods and Bishop 2011).
Mouse DCT is around 60 kb, and contains eight exons. An Mbox located at -150 bp to -140 bp of mouse DCT is the transcription initiation site, while the equivalent in human DCT is located at -135 bp to -125 bp. TYR and TYRP1 mouse promoters also have M-boxes at similar positions (Budd and Jackson 1995). In humans, a 32-bp element spanning from -268 bp to -56 bp and a proximal region located at -447 bp to -415 bp are necessary for pigment cell-specific expression of DCT (Yokoyama et al. 1994). Moreover, melanogenesis and melanocyte cellular differentiation are regulated by the microphtalmiaassociated transcription factor (MITF) and sex determining region Y-box 10 (SOX10), which have been verified as transcription factors that bind the DCT promoter (Ludwig et al. 2004).
Based on existing knowledge of the DCT promoter in mice and humans, we hypothesized that an optimal melanin cell-specific DCT promoter for sheep skin melanocytes could be developed. To obtain an efficient DCT promoter region for use in sheep skin, five segments of the DCT promoter were cloned, analyzed, inserted into eukaryotic expression vectors, and transfected into sheep melanocytes. Our results indicated that the region -593 bp to + 41 bp is the most efficient for expression in sheep skin melanocytes.

Sample collection
All animal procedures followed the Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments. Five healthy 2-year-old small fat-tailed sheep were selected for sample collection from a sheep farm in Jiexiu, Shanxi Province, China. Five pieces of skin (10 × 10 mm 2 ) from the ears were obtained through punch skin biopsy under local anesthesia and immediately stored in liquid nitrogen.

Sequence analysis
BLAST sequence analysis (https://www.ncbi.nlm.nih.gov/) was used to analyze sequence homology with reference sequences. The motifs of the DCT promoter were predicted by Primer 5.0 software based on the longest fragment.

Sheep skin melanocyte culture and transfection
Sheep skin melanocytes were donated by the Laboratory of Alpaca Biology, Shanxi Agricultural University, China, and cultured in MelM medium (ScienCell Research Laboratories, Carlsbad, CA, USA).
Based on the manufacturer's instructions, eight groups with different ratios of plasmids (pMSCV PIG vector (Addgene, Cambridge, MA, USA)) and transfection reagents  were incubated with melanocytes in 24-well plates ( Table 2). The medium was replaced with fresh serum-free medium (MelM) 6 h after transfection, then cells were incubated for an additional 48 h. GFP (Green Fluorescent Protein) expression was then observed using an inversion fluorescence microscope (Leica, Wetzlar, Germany) to select the optimal volume of plasmids and transfection reagents.
Based on these optimization conditions, five recombinant plasmids (441 bp, 652 bp, 834 bp, 1155 bp, and 1504 bp) were transfected into sheep skin melanocytes using the X-treme-GENE HP DNA Transfection Reagent (Roche, Basel, Switzerland). pMSCV PIG vector was used as control. Forty-eight to 72 h after transfection, GFP expression was observed and measured by Image Pro Plus 6.0 software. Each experiment was repeated three times for each group and three fields were selected to detect GFP expressions.

Statistical analysis
Differences in GFP expression among melanocytes transfected with different DCT promoter fragments were analyzed using SPSS software.

Amplification of sheep DCT promoter fragments
PCR amplification produced five fragments that were about 441 bp, 652 bp, 834 bp, 1155 bp, and 1504 bp in length (Figure 2).
Primer 5 software identified motifs within the 1504 bp fragment, including a CAAT box, M-box, and E-box (Table 3; Figure 3).

The optimal ratio of plasmid to transfection reagents
GFP expression was highest when the ratios of plasmids to transfection reagents were 2:3 and 2:4 ( Figure 5). Taking the cytotoxicity of transfection reagents into account, we selected the ratio of 2:3 for further experiments. This corresponded to 1 μg plasmid and1.5 μL transfection reagent per well of a 24-well plate.

Expression activity of sheep DCT promoter
GFP expression varied among the different transfection groups, with the following trend: 652 bp > 834 bp > 441 bp > 1504 bp > 1155 bp. GFP expression in the 1155 bp group was significantly lower than in the 652 bp and 834 bp groups (p < 0.01), while GFP expression in the 1504 bp group was significantly lower than in the 652 bp group (p < 0.05) ( Figure 6). This was observed from the analysis of fluorescent images (Figure 7). These findings indicated that the entire DCT promoter region from -1247 bp to + 237 bp can initiate transcription, but that the optimal active region is located between -593 bp and + 41 bp.

Discussion
In the present study, we showed that the sheep DCT promoter contains a CAAT box, M-box, and E-box. The CAAT box signals Table 2. Volume of DNA and transfection reagents used in cell transfection.

Ratio
Plasmid (μg) Transfection reagents (μL) 1:3 0.5 1.5 1:4 0.5 2 1:5 0.5 2.5 1:6 0.5 3 2:3 1 1.5 2:4 1 2 2:5 1 2.5 2:6 1 3 Figure 2. PCR products detected by agarose gel electrophoresis. Marker: DL2000 marker; lanes 1 and 2: 441 bp PCR product; lanes 3 and 4: 652 bp PCR product; lanes 5 and 6: 834 bp PCR product; lanes 7 and 8: 1155 bp PCR product; lanes 9 and 10: 1504 bp PCR product.  the binding site for RNA transcription factors, and is typically accompanied by a conserved consensus sequence. Its function is to enable gene transcription at sufficient quantities (Dolfini et al. 2009). The M-box is the most striking feature of the promoter region of TYR family members, and is located between -160 bp and -40 bp. We found that the sheep DCT M-box was located at -139 bp to -129 bp upstream of the transcription start site, which is similar to the location in human DCT (Budd and Jackson 1995). The M-box is the binding site of MITF, and the SOX10 binding site is located in its immediate vicinity (Ludwig et al. 2004). MITF, a basic helix-loop-helix leucine zipper transcription factor, regulates melanocyte cellular differentiation and the transcription of melanogenic enzymes such as TYR, TYRP1, and DCT (Levy et al. 2006). SOX10 has been shown to regulate MITF expression in melanocytes (Britsch et al. 2001), although dog DCT lacks SOX10 binding sites Kowalczyk et al. 2016). The E-box binds transcription factors to imitate transcription (Chaudhary and Skinner 1999), and the E-box of TYR family promoters is also the binding site of MITF (Miccadei et al. 2008).
A previous study of human DCT revealed two regulatory regions, a 32-bp element from -447 bp to -415 bp and the proximal region from -268 bp to -56 bp, that are required for pigment cell-specific expression (Yokoyama et al. 1994). However, we did not find similar regions in the sheep DCT promoter. Instead, we identified E-box within the two regulatory regions.
GFP expression did not differ significantly different after transfection of 441 bp, 652 bp, and 834 bp fragments of the DCT promoter, especially between 652 bp and 834 bp groups. This indicated that -593 bp to + 41 bp is the optimal region of the DCT promoter, and that the region between -593 bp and -396 bp might contain positive regulatory elements. GFP expression in cells transfected with the 1155 bp fragment was significantly lower than in the 652 bp and 834 bp groups, suggesting that positive regulatory elements may exist between -929 bp and -789 bp or between + 41 bp and + 206 bp. However, GFP expression in cells transfected with the 1504 bp fragment was higher than in the 1155 bp group, indicating the possibility of positive regulatory elements between -1247 bp and -929 bp or between + 206 bp and + 237 bp. . GFP expression in different transfection groups. Highest and lowest GFP expression occurred in the 652 bp group and 1155 bp group, respectively. GFP expression in the 1155 bp group was significantly lower than in the 652 bp and 834 bp groups (p < 0.01). GFP expression in the 1504 bp group was significantly lower than in the 652 bp group (p < 0.05). Each experiment was repeated three times for each group. Values represent the mean ± SE from three independent experiments. * significant difference, ** extremely significant difference. Figure 5. Transfection efficiencies with different ratios of plasmids and transfection reagents. The efficiency increased when the ratio increased from 1:3 to 1:6, and was highest at ratios of 2:3 and 2:4. The efficiency decreased with excess transfection reagents.
The shortest fragment of 441 bp (-396 bp to + 27 bp) initiated transcription in sheep skin melanocytes. This compares with minimal regions of just 150 bp upstream of the initiation codon for the melanocortin receptor 1 promoter (Miccadei et al. 2008), 270 bp upstream of the transcription start site for the TYR promoter (Kluppel et al. 1991), and between -44 bp to + 107 bp for the TYRP1 promoter (Lowings et al. 1992).

Conclusion
The entire region of the sheep DCT promoter from -1247 bp to + 237 bp can initiate transcription, but the optimal active region is located between -593 bp and + 41 bp.