Abstract：Bone sialoprotein (BSP) is a major component of medullary bone in chicken (Gallus gallus) and plays an important role in bone remodeling and mineralization. To explore the regulatory mechanism of BSP gene expression and the effect of variations in the promoter region on gene expression in laying hens,Hyline Grey laying hens were used as experimental animals,and the sequence variation in BSP promoter region was analyzed with gene cloning and sequencing technology. The online software JASPAR2018 was used for binding site prediction of transcription factors. Luciferase reporter gene recombinant vectors with different length of promoter fragments were constructed and transfected into chicken fibroblast line DF-1 whose dual-luciferase activity was measured. The expression of BSP gene was determined by qRT-PCR method. The results showed that there were 6 haplotypes (Hap1~6) in BSP promoter (-1257~+142 bp),and the core promoter region was located at -202~+142 bp. The difference of transcriptional activity was also found between Hap1 and Hap5 in the promoter region. The high transcriptional activity of Hap1 in the core promoter region caused an increase in the expression level of BSP gene. In conclusion,the core region in promoter of chicken BSP gene was identified and cloned successfully,which could provide basic materials for the investigation of regulatory mechanism of BSP gene expression and genetic selection of bone quality in laying hens.
[1] 陈伟健,晋大祥,谢炜星,等.2018.RUNX2基因参与骨代谢相关通路的研究进展[J].中国骨质疏松杂志,24(4):557-560. (Chen W J,Jin D X,Xie W X,et al.2018.Advance in the research ofRUNX2gene in bone metabolism-related pathway[J].Chinese Journal of Osteoporosis,24(4):557-560.) [2] 李彬,张柳.2009.RUNX2与骨代谢的调控[J].中国骨质疏松杂志,15(1):63-67. (Li B,Zhang L.2009.RUNX2 and bone metabolism regulation[J].Chinese Journal of Osteoporosis,15(1):63-67.) [3] 李丛华,董伟,吴亚菲.2005.骨涎蛋白在组织矿化中的作用[J].国外医学口腔医学分册,32(6):449-451. (Li C H,Dong W,Wu Y F.2005.The role of bone sialoprotein in tissue mineralization[J].International Journal of Stomatology,32(6):449-451. [4] 苏尚,吴畏.2014.Wnt/β-catenin信号通路对靶基因转录的调控[J].中国科学:生命科学,44(10):1029-1042. (Su S,Wu W.2014.Regulation of target gene transcription by Wnt/β-catenin signaling[J].Scientia Sinica (Vitae),44(10):1029-1042.) [5] 袁林,曹凯华.2012.骨涎蛋白在不同类型种植体骨界面改建中的变化[J].童心牙体牙髓牙周病学杂志,22(6):319-322. (Yuan L,Cao K H.2012.Expression of bone sialoprotein in the process of implant-bone interface remodeling with different implants[J].Chinese Journal of Conservative Dentistry,22(6):319-322.) [6] 张小霞,张颖,辛晓燕.2017.EGR转录因子在肿瘤中的研究进展[J].现代肿瘤学,25(04):654-657. (Zhang X X,Zhang Y,Xin X Y.2017.The latest research progress of EGR transcription factors in the tumors[J].Journal of Modern Oncology,25(04):654-657.) [7] 郑炜,马俊福,朱跃兰,等.2018.转录因子Egr2/Egr3的生物学作用及与自身免疫性疾病的相关性[J].医学研究生学报,31(5):524-528. (Zheng W,Ma J F,Zhu Y L,et al.2018.Study on Egr2/Egr3 and autoimmune diseases[J].Journal of Medical Postgraduates,31(5):524-528.) [8] Bauer O,Sharir A,Kimura A,et al.2015.Loss of osteoblast Runx3 produces severe congenital osteopenia[J].Molecular and Cellular Biology,35(7):1097-1109. [9] Boudiffa M,Wade-Gueye N M,Guignandon A,et al.2010.Bone sialoprotein deficiency impairs osteoclastogenesis and mineral resorptionin vitro[J].Journal of Bone and Mineral Research,25(12):2669-2679. [10] Buckland J.2015.Anabolic Wnt/β-catenin signaling:Osteocytes are key[J].Nature Reviews Rheumatology,11(3):128. [11] Ducy P,Starbuck M,Priemel M,et al.1999.A Cbfal-dependent genetic pathway controls bond formation beyond embryonic development[J].Genes Development,13(8):1025-1036. [12] Fischbach N A,Rozenfeld S,Shen W,et al.2005.HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursorin vitroand causes hematopoietic stem cell expansion and acute myeloid leukemiain vivo[J].Blood,105(4):1456-1466. [13] Fisher L W,Fedarko N S.2003.Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins[J].Connective Tissue Research,44(1):33-40. [14] Fisher L W,Torchia D A,Fohr B,et al.2001.Flexible structures of SIBLING proteins,bone sialoprotein,and osteopontin[J].Biochemical and Biophysical Research Communications,280(2):460-465. [15] Fukasawa K,Park G,Iezaki T,et al.2016.ATF3 controls proliferation of osteoclast precursor and bone remodeling[J].Scientific Reports,6(1):30918. [16] Gabet Y,Baniwal S K,Leclerc N,et al.2010.Krox20/EGR2 deficiency accelerates cell growth and differentiation in the monocytic lineage and decreases bone mass[J].Blood,116(19):3964-3971. [17] Hunter G K,Goldberg H A.1994.Modulation of crystal formation by bone phosphoproteins:Role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein[J].Biochemical Journal,302(1):175-179. [18] Jeong B C,Kim J H,Kim K,et al.2017.ATF3 modulates calcium signaling in osteoclast differentiation and activity by associating with c-Fos and NFATc1 proteins[J].Bone,95:33-40. [19] Ji C,Liu X H,Xu L,et al.2017.RUNX1 plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal stem cells line C3H10T1/2,murine multi-lineage cells lines C2C12 and MEFs[J].International Journal of Molecular Sciences,18(7):e1348. [20] Jill A H,Maria H G,Kim W K,et al.2016.Bone sialoprotein keratan sulfate proteoglycan (BSP-KSPG) and FGF-23 are important physiological components of medullary bone[J].Comparative Biochemistry and Physiology,Part A,194:1-7. [21] Kayvan Z,Cassie J L,Leonard G.2017.SIRT1 is a positive regulator of the master osteoblast transcription factor,RUNX2[J].PLOS ONE,12(5):e0178520. [22] Kobayashi Y,Uehara S,Udagawa N,et al.2016.Regulation of bone metabolism by Wnt signals[J].The Journal of Biochemistry,159(4):387-392. [23] Komaki M,Asakura A,Rudnicki M,et al.2004.MyoD enhances BMP7-induced osteogenic differentiation of myogenic cell cultures[J].Journal of Cell Science,117(8):1457-1468 [24] Kruger T E,Miller A H,Godwin A K,et al.2014.Bone sialoprotein and osteopontin in bone metastasis of osteotropic cancers[J].Critical Reviews in Oncology Hematology,89(2):330-341. [25] Li Z Q,Xu Z M,Duan C C,et al.2018.Role of TCF/LEF transcription factors in bone development and osteogenesis[J].International Journal of Medical Sciences,15(12):1415-1422. [26] Lu K,Shi T S,Shen S Y,et al.2018.Egr1 deficiency disrupts dynamic equilibrium of chondrocyte extracellular matrix through PPARγ/RUNX2 signaling pathways[J].American Journal of Translational Research,10:1620-1632. [27] Malaval L,Wade-Guéye N M,Boudiffa M,et al.2008.Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis[J].Journal of Experimental Medicine,205(5):1145-1153. [28] Masi L,Brandi M L,Robey P G,et al.1995.Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1)[J].Journal of Bone and Mineral Research,10(2):187-196. [29] Merrill B J,Pasolli H A,Polak L,et al.2004.Tcf3:A transcriptional regulator of axis induction in the early embryo[J].Development,131(2):263-274. [30] Michikami I,Fukushi T,Tanaka M,et al.2012.Krüppel-like factor 4 regulates membranous and endochondral ossification[J].Experimental Cell Research,318(4):311-325. [31] Sabag E,Halperin E,Liron T,et al.2019.Hormone-independent sexual dimorphism in the regulation of bone resorption by Krox20[J].Journal of Bone Mineral Research,34(12):2277-2286. [32] Soenjaya Y,Foster B L,Nociti F H,et al.2015.Mechanical forces exacerbate periodontal defects in Bsp-null mice[J].Journal of Dental Research,94(9):1276-1285. [33] Tang C,Chen W,Luo Y,et al.2020.Runx1 up-regulates chondrocyte to osteoblast lineage commitment and promotes bone formation by enhancing both chondrogenesis and osteogenesis[J].Biochemical Journal.DOI:10.1042/BCJ20200036. [34] Toshihisa K.2015.The functions of Runx family transcription factors and Cbfb in skeletal development[J].Oral Science International,12(1):1-4. [35] Wang Y F,Feng Q L,Ji C X,et al.2017.RUNX3 plays an important role in mediating the BMP9-induced osteogenic differentiation of mesenchymal stem cells[J].International Journal of Molecular Medicine,40(6):1991-1999. [36] Yu M,Zhan J,Zhang H Q.2020.HOX family transcription factors:Related signaling pathways and post-translational modifications in cancer[J].Cellular Signalling,66:109469.
