Identifycation Of SALL4 Alternative Splicings And Their Function To Regulate OTX2 Expression

Sall4(Spalt-like transcription factor 4) is a kind of C2H2 zinc finger protein transcription factors, playing an important role on the establishment of stem cell lines and maintain pluripotency. Sall4 can be widely expressed in tissues. It has multiple isoformswhich has been published two isoforms Salll4 A and Sall4 B in person(Ma et al., 2006) and mouse(Rao et al., 2010). Sall4 B missing most of the second exoncompared with Sall4 A and it plays a very important role in maintain pluripotency of stem cell. Otx2(Orthodenticle Homeobox 2) belongs Otx transcription factor superfamily(Finkelstein et al, 1990;. Simeone et al, 1992; Simeone et al, 1993), The family played an important role in the early development of photoreceptor cells andthe medulla oblongata of the brain(Matsui et al, 1989; Gronwald et al, 1988.). It has been reported thatmouse Otx2 portein involved in the regulation oftransition between Naive state PSCs and primed state PSCs, which plays a vital role on balance self-renewal and differentiation potential in mouse embryonic stem cells(embryonic stem cells, ESCs)(Dario et al., 2012). Our study was intend to clone pig SALL4 two isoforms SALL4 A and SALL4 B, and study the direct regulation of SALL4 isoforms on the OTX2. The study prove that the pig SALL4 existence different isoforms, furthermore we can enrich that Sall4 directly regulate OTX2 in stem cell network. This study can lay the foundation for the further study of the reprogramming control mechanism of pluripotent stem cells.Main conclusions obtained in this study were:1. Cloning and functional verification of pig SALL4 different isoforms3153 bp and 1851 bp of the SALL4 A and SALL4 B were cloned from porcine iPS, and constructed pEGFP-SALL4 A and pEGFP-SALL4 B reporter vector and fusion protein were identification with westernblot. Repoter vectors were transfected to 293 T cells, fusion protein expression location were observed byfluorescence microscope. We detect the expression of SALL4 in different tissues(brain, spine, skin, heart, muscle, spleen, kidney, thymus, fat, testis, ovary, liver), different cells(PEF, iPS-j, iPS-g and iPS-w) by quantitative PCR, which indicate that SALL4 gene is highly expressed only in the testis, ovary and iPSCs with tissuesspecific.2. Cloning, screening core regulation region and functional verification of SALL4 promoter2123 bp promoter sequences were cloned from kidney tissues of pigs, and then the sequences were connected to pEGFP-1 and p GL3-Basic vectors. Reporter vectors were transfected to P19、293T、CHO、Hela and PK15 cells and the results showed that pig SALL4 gene promoter was activated in P19、293T、CHO and Hela cells but not in PK15 cells with tissue specificity. Bioinformatics was used for analyzing this promoter sequence, which showed the potential transcription factor binding sites of OTX2, OCT4, SOX2, KLF4, ESRRA and ESRRB etc. SALL4 promoter and these transcription factors were co-transfected in 293 T cells, and we detection the promoter activityusing the dual luciferase assay. The experimental results show OCT4, SOX2, C-Myc, ESRRA, ESRRB, SMAD2, SMAD3 can promoting SALL4 promoter in varying degrees. To study the regulatory features of different regions in SALL4 gene promoter, different fragments(1901 bp, 1071 bp, 532 bp, 176 bp) of SALL4 gene promoter were cloned and connected to pGL3-Basic vectors respectively in this experiment. Then these reporter vectors were used to transfect 293 T cells, the results of the dual-luciferase essays showed that the core promoter area was located within-368 bp to-851 bp in the 5’ flanking sequence.3. The interaction between the two isoforms of SALL4 and pig OTX2Potential OTX2 binding sites were predicted on pig SALL4 promoter. We test theexpression of SALL4 by real-time quantitative PCR and WB in pig iPS cells. SALL4 A and SALL4 B expression level were reduced through the cells overexpressing of OTX2, but SALL4 A and SALL4 B expression level were increased through the cells knockdown of OTX2, which indicating that OTX2 inhibit the expression of SALL4. The different lengths of SALL4 promoter and pcDNA-OTX2 were co-transfected in to 293 T cells. SALL4 promoter activitywas detected using the dual luciferase reporter system. Experimental results show that there is a suppression regulatory sites of OTX2 in the region of-368 bp to-851 bp in SALL4 promoter.In order to prove the SALL4 regulate the expression of OTX2, different fragments(O-1865 bp, O-1450 bp, O-1038 bp, O-203 bp) of OTX2 gene promoter were cloned and connected to pGL3-Basic vectors. The different lengths of OTX2 promoter and pMX-SALL4 were co-transfected into 293 T cells, OTX2 promoter activity was detectedusing the dual luciferase reporter system. Experimental results show that there is a suppression regulatory sites of SALL4 in the region of-915 bp to-1327 bp in OTX2 promoter.The experimental results confirmed the prophase research work in our lab, and shows that overexpression OTX2 in the pig iPS cells, lead the shape of iPS cells loosing, AP staining uneven, AP positive clone number decreased significantly. Furthermore, when overexpression of SALL4 A or SALL4 B in OTX2+ cells, the iPS clone shape has improved, AP stained uniformly, AP positive clones increased, which shown that SALL4 inhibit the expression of OTX2.