Rb1基因启动子和增强子的鉴定

The identification of the promoter and enhancer of Rb1 gene

视网膜母细胞瘤易感基因(retinoblastoma susceptibility gene)为癌变过程中的重要肿瘤抑制基因.该基因参与调控体内多个生理过程,主要包括细胞周期、细胞凋亡以及细胞分化等进程.Rb1主要通过与E2F1/DP1复合物的相互作用调控细胞周期运转和细胞凋亡过程.早期小鼠遗传学研究表明,Rb1基因缺失会导致骨骼肌细胞凋亡.进一步的研究表明,Rb1也能调节肌源调控因子(myogenic regulatory factors,MRFs)的转录活性,从而促进骨骼肌细胞分化.然而目前关于Rb1基因在骨骼肌发育过程中的表达调控机制尚未清晰.本文通过生物信息学分析、转基因小鼠模型、转录分析以及突变体分析等方法,初步鉴定了Rb1基因在骨骼肌中表达调控的启动子和增强子.结果表明:(1)生物信息学鉴定出的启动子和增强子之间存在明显的协同效应;(2)Rb1基因的启动子和增强子可以驱动LacZ报告基因在胚胎体节和成体骨骼肌组织中的表达;(3)启动子和增强子序列中保守E-box位点的突变会导致报告基因活性急剧下降;(4)MRFs能够激活Rb1基因启动子和增强子驱动的报告基因.这些研究发现有助于更全面地了解Rb1基因的转录调控.

The retinoblastoma susceptibility gene(Rb1)is an important tumor suppressor gene during tumorigenesis,which is also involved in multiple physiological processes,including cell cycle,cellular apoptosis and cell differentiation.The interaction between Rb1 and E2F1/DP1 plays a pivotal role in the cell cycle regulation and cellular apoptosis.The essential role of Rb1 gene in the skeletal muscle has been demonstrated in the Rb1 null mice,in which the myogenic cells went to apoptosis during embryonic development.Biochemical studies have also shown that Rb1 may regulate the transcription activity of myogenic transcription factors(MRFs),thereby promoting myogenic differentiation.MRFs(including MyoD,Myf5,Myf6 and Myog)belong to a superfamily of basic helix-loop-helix(bHLH)transcription factor,which heterodimerize with E proteins and bind to the specific DNA sequence as E-box(CANNTG).Extensive studies have revealed the essential roles of MRFs in the skeletal muscle development,and identified a broad range of downstream skeletal muscle-specific genes.However,it is still not well understood about the mechanisms of the transcriptional regulation of Rb1 during skeletal muscle development.Previous study has reported the murine Rb16 kb promoter,which directed the transgenic reporter expression exclusively to the central and peripheral nervous system,while the transgenic reporter lines were unable to recapitulate Rb1 expression in the somite during murine embryonic development.In the present studies,we have utilized a number of techniques,including bioinformatic analysis,transgenic mice model,transcriptional assays,and mutagenic analysis to investigate the regulatory mechanisms of Rb1 gene expression.Here,we report several interesting discoveries:(1)We have identified the 1.6 kb promoter and 1.7 kb enhancer of the Rb1 gene through bioinformatics analysis.Our further studies have shown the significant synergistic interaction between the promoter and enhancer through transcriptional assays.(2)We have constructed the transgenic reporter vector(1.6 kb promoter-Hsp68-LacZ-1.7 kb enhancer)and generated transgenic Rb1-LacZ mouse model through the service of the GemPharmatech Company.Two transgenic Rb1-LacZ reporter lines have been established successfully:#7 and#10.The following LacZ staining analysis has confirmed the LacZ reporter expression in the embryonic somite and adult skeletal muscle(gastrocnemius muscle,soleus,tibialis anterior and extensor digitorum longus).These transgenic studies have demonstrated that the 1.6 kb promoter and 1.7 kb enhancer of Rb1 is sufficient to drive the LacZ reporter expression in the somite and adult skeletal muscle.(3)We have examined the functional roles of the four conserved E-box motifs in the 1.6 kb promoter and 1.7 kb enhancer of Rb1 through mutagenesis.Our preliminary studies have revealed that the mutation of these four E-box sites resulted in a significant decrease of the reporter activity during myogenic differentiation.(4)Transcriptional assays have shown that MRFs can transactivate the luciferase reporter harboring the 1.6 kb promoter and 1.7 kb enhancer of Rb1 gene through the NIH 3T3 cells.In summary,our studies have uncovered another regulatory cascade MRFs-Rb1-E2F1/DP1 in the cell apoptosis during skeletal muscle development.