MITF基因突变致Ⅱ型Waardenburg综合征发病的实验研究

Experimental Studies on Pathogenesis of Waardenburg Syndrome TypeⅡ Resulting from Microphthalmia-Associated Transcription Factor Gene Mutations

张华;陈红胜;李家大;罗洪金;梅凌云;贺楚峰;冯永;

1:新疆医科大学第一附属医院耳鼻咽喉科

2:中南大学湘雅医院耳鼻咽喉科

3:中南大学医学遗传学国家重点实验室

摘要
目的通过体外实验研究小眼球畸形相关转录因子(microphthalmia-associated transcription factor,MITF)突变基因功能,初步探讨其致Ⅱ型Waardenburg综合征(Waardenburg syndrome,WS)发病的分子机制。方法以野生型MITF基因真核细胞表达质粒pCMV-MITF-Flag为模板分别构建二个致Ⅱ型WS的MITF基因新发突变R217I和T192fsX18的真核细胞表达质粒。野生MITF和突变R217I和T192fsX18表达质粒瞬时转染黑色素瘤细胞或小鼠胚胎成纤维细胞(NIH3T3细胞),应用Western blot和细胞免疫荧光分别检测其表达和亚细胞定位;应用荧光素酶活性检测系统通过对酪氨酸酶(tyrosinase,TYR)报告基因活性检测观察野生/突变MITF蛋白对其靶基因TYR转录活性的调控作用,以及二个突变蛋白对野生MITF蛋白功能的影响;应用生物素标记的含E box基序(CATGTG)的DNA寡核苷酸链探针分别沉淀MITF、R217I及T192fsX18蛋白,检测野生/突变MITF蛋白与靶基因TYR启动子的结合力。结果成功构建了突变型R217I、T192fsX18真核细胞重组表达质粒pCMV-R217I-Flag和pCMV-T192fsX18-Flag,MITF蛋白与R217I、T192fsX18突变蛋白在黑色素瘤细胞中正确表达,进一步验证了重组质粒构建的正确性。突变R217I蛋白与野生MITF蛋白一样仅在细胞核中分布,而T192fsX18蛋白则出现异常亚细胞定位,仅在细胞质中分布。尽管R217I蛋白仍残余部分功能可增加TYR启动子转录活性,但与野生MITF蛋白相比,二者差异有显著统计学意义(P<0.01),而T192fsX18蛋白则完全失去调控TYR启动子转录活性作用(P<0.01);二者均未对野生MITF蛋白功能产生显性负效应(P>0.05)。突变R217I蛋白与野生MITF蛋白均可与TYR启动子特异DNA序列E-box结合,而突变T192fs X18蛋白则不能与之结合。结论 R217I和T192fsX18突变蛋白通过影响靶基因TYR转录活性,使其表达下调、黑色素合成减少,以单倍体剂量不足效应致Ⅱ型WS。
关键词
Waardenburg综合征;小眼球畸形相关转录因子;基因突变;体外实验
基金项目(Foundation):
国家自然科学基金(81260160,81170923)资助
作者
张华;陈红胜;李家大;罗洪金;梅凌云;贺楚峰;冯永;
参考文献

1 Nayak CS,Isaacson G.Worldwide distribution of Waardenburg syndrome[J].Ann Otol Rhinol Laryngol,2003,112:817.

2 Sauka-Spengler T,Bronner-Fraser M.Development and evolution of the migratory neural crest:agene regulatory perspective[J].Curr Opin Genet Dev,2006,16:360.

3 Thomas AJ,Erickson CA.FOXD3regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism[J].Development,2009,136:1 849.

4 Pingault V,Ente D,Dastot-Le Moal F,et al.Review and update of mutations causing Waardenburg syndrome[J].Hum Mutat,2010,31:391.

5 Mosammaparast N,Pemberton L.Karyopherins:from nuclear-transport mediators to nuclear-function regulators[J].Trends Cell Biol,2004,14:547.

6 Hou L,Pavan WJ.Transcriptional and signaling regulation in neural crest stem cell-derived melanocyte development:do all roads lead to Mitf?[J].Cell Res,2008,18:1 163.

7 Bejar J,Hong Y,Schartl M.Mitf expression is sufficient to direct differentiation of medaka blastula derived stem cells to melanocytes[J].Development,2003,130:6 545.

8 McGill GG,Horstmann M,Widlund HR,et al.Bcl2regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability[J].Cell,2002,109:707.

9 Lowings P,Yavuzer U,Goding CR.Positive and negative elements regulate a melanocyte-specific promoter[J].Mol Cell Biol,1992,12:3 653.

10 Bentley NJ,Eisen T,Goding CR.Melanocyte-specific expression of the human tyrosinase promoter:activation by the microphthalmia gene product and role of the initiator[J].Mol Cell Biol,1994,14:7 996.

11 Bertolotto C,Busca R,Abbe P,et al.Different cis-acting elements are involved in the regulation of TRP1and TRP2promoter activities by cyclic AMP:pivotal role of M boxes(GTCATGTGCT)and of microphthalmia[J].Mol Cell Biol,1998,18:694.

12 Yavuzer U,Keenan E,Lowings P,et al.The Microphthalmia gene product interacts with the retinoblastoma protein in vitro and is a target for deregulation of melanocyte-specific transcription[J].Oncogene,1995,10:123.

13 Yasumoto K,Yokoyama K,Shibata K,et al.Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene[J].Mol Cell Biol,1994,14:8 058.

14 Tassabehji M,Newton VE,Read AP.Waardenburg syndrome type 2caused by mutations in the human microphthalmia(MITF)gene[J].Nat Genet,1994,8:251.

15 Nobukuni Y,Watanabe A,Takeda K,et al.Analyses of loss-of-function mutations of the MITF gene suggest that haploinsufficiency is a cause of Waardenburg syndrome type 2A[J].Am J Hum Genet,1996,59:76.

16 Schwarzbraun T,Ofner L,Gillessen-Kaesbach G,et al.A new 3p interstitial deletion including the entire MITF gene causes a variation of Tietz/Waardenburg type IIA syndromes[J].Am J Med Genet A,2007,143:619.

17 Takebayashi K,Chida K,Tsukamoto I,et al.The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential[J].Mol Cell Biol,1996,16:1 203.

18 Takeda K,Takemoto C,Kobayashi I,et al.Ser298of MITF,a mutation site in Waardenburg syndrome type 2,is a phosphorylation site with functional significance[J].Hum Mol Genet,2000,9:125.

19 Chen H,Jiang L,Xie Z,et al.Novel mutations of PAX3,MITF,and SOX10genes in Chinese patients with type I or type II Waardenburg syndrome[J].Biochem Biophys Res Commun,2010,397:70.

20 Meadows NA,Sharma SM,Faulkner GJ,et al.The expression of Clcn7and Ostm1in osteoclasts is coregulated by microphthalmia transcription factor[J].J Biol Chem,2007,282:1 891.

21 Arnheiter H.The discovery of the microphthalmia locus and its gene,Mitf[J].Pigment Cell Melanoma Res,2010,23:729.

22 Cheli Y,Ohanna M,Ballotti R,et al.Fifteen-year quest for microphthalmia-associated transcription factor target genes[J].Pigment Cell Melanoma Res,2010,23:27.

23 Vachtenheim J,Novotna H,Ghanem G.Transcriptional repression of the microphthalmia gene in melanoma cells correlates with the unresponsiveness of target genes to ectopic microphthalmia-associated transcription factor[J].J Invest Dermatol,2001,117:1 505.

24 Tachibana M.Evidence to suggest that expression of MITF induces melanocyte differentiation and haploinsufficiency of MITF causes Waardenburg syndrome type 2A[J].Pigment Cell Res,1997,10:25.

25 Kim DK,Morii E,Ogihara H,et al.Different effect of various mutant MITF encoded by mi,Mior,or Miwh allele on phenotype of murine mast cells[J].Blood,1999,93:4 179.