The Molecular Mechanism Of SOX6 Regulating Pigmentation In Alpaca Melanocytes

Melanin pigmentation mainly includes melanin production and transport,which were regulated by many genes.SOX6 is a novel transcriptional factor in melanocytes,but its mechanism has not been reported.In this present research,the expression and function of SOX6 in alpaca melanocytes were investigated.The methods and results are as follows:1.The cloning and sequence analysis of SOX6 gene revealed that the conserved functional domain of this protein included SOX-T cytokine.The amino acid T119 in the MTSVTFGTPERRKGS sequence motif may be phosphorylated by CDK5.The sequence of the phosphorylation site of alpaca SOX6 protein shared >90% of human,dog,horse,sheep and rat,and at the predicted phosphorylation site,the alpaca sequence had the highest similarity with the corresponding bovine sequence.2 CDK5 is a protein kinase that regulates melanin production and may also play an important role in the transport process.Therefore,in this research,SOX6 was overexpressed and inhibited in alpaca melanocytes,and the function of SOX6 in melanin formation was verified by q PCR,western blotting and other methods.The results of cell immunohistochemistry showed that SOX6 was expressed in the cytoplasm of alpaca melanocytes.SOX6 interacts with cyclin-dependent kinases CDK5,β-catenin and Cyclin D1.SOX6 overexpression upregulated the m RNA expression and protein level expression of Cyclin D1,CDK5,MITF,TYR,TYRP1 and DCT,decreased the m RNA expression and protein level expression of β-catenin,and vice versa.The over-expression of SOX6 increased melanin production in alpaca melanocytes,which suggested that SOX6 played a regulatory role in melanin production in alpaca melanocytes.3.In order to further reveal whether SOX6 plays a regulatory role in melanin transport,here,the target genes of SOX6 were mined by immunoprecipitation(Co-IP)combined with Ch IP-seq method.The results showed that SOX6 directly targeted and regulated many genes,including MLPH,an important regulatory gene for melanin transport,and MAP3K4,MYO5 b,MLPH,TMEM63 A and SLC6A1,which are important genes in the pathway of melanin production.The q PCR results showed that SOX6 increased the m RNA expression levels of MAP3K4,MYO5 b,MLPH,TMEM63 A and SLC6A1 genes.4.In order to screen the strongest promoter region of SOX6 on MLPH transcription,the double luciferase reporter gene was used to screen the strongest promoter region of MLPH.The results showed that the MLPH promoter region,divided into four segments,of which the second segment(F2)was the most active.q PCR and western blotting results showed that the expressions of MLPH,MYO5 a and Rab27 a were increased.Fontana-Masson staining results showed that the transcription factor SOX6 increased the production of melanin in mealnocytes.Transmission electron microscope(TCM)results showed that compared to the NC group,the endoplasmic reticulum in melanocytes with SOX6 over expression was significantly increased,melanosomes at different stages appeared,and the microfilament and microtubules were also significantly increased.5.In order to reveal the downstream molecular pathways and biological processes affected by SOX6 in regulating the biological process of melanocytes,the lnc RNA(named FBXO22-ASI)interacting with MLPH predicted by biological information was constructed as an overexpression vector,and transferred to alpaca melanocytes for transcriptome sequencing.The results showed that FBXO22-ASI was involved in the regulation of multiple molecular pathways,including autophagy pathway,MAPK pathway,and Wnt signaling pathway.q PCR analysis of m RNA expression levels of ATG5,MAPK3,STMN1,TSPAN12 and MITF showed that the results were consistent with the results of RNA sequencing.Conclusion: transcription factor SOX6 affects melanin production and transport in melanocytes by phosphorylation by CDK5 and targeting MLPH to regulate the expression of key melanin production genes MITF,TYR,TYRP1 and TYRP2 as well as the transporter genes MYO5 a and Rab27 a,thereby regulating melanin pigmentation in alpaca melanocytes.At the same time,it is also involved in autophagy and other pathways in melanocytes,which may play an important role in other biological functions,and provide theoretical basis for subsequent studies on SOX6 function.