HCF-1和NSPc-1调控细胞周期的分子机制研究

【作者】 岳继平；

【导师】 强伯勤； 袁建刚； 彭小忠；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 宿主细胞因子HCF-1(Host Cell Factor-1)是细胞增殖过程中必需的染色质相关蛋白，在细胞周期调控中发挥重要的作用。哺乳动物细胞中敲低该基因表达水平后，大部分细胞都会被阻滞在G1期，并且细胞的正常有丝分裂也会受到影响，出现多核现象(multi-nucleation)。Cdc42作为Rho GTPase家族蛋白中的重要成员，也具有类似的细胞周期调控作用。NSPc-1是2001年才被报道发现的PcG家族新成员，该基因存在于成人的所有组织中。前期研究工作发现，NSPc-1作为一个典型的PcG家族蛋白，具有核转录抑制因子的特性，并且NSPc-1对细胞G1-S期进程具有一定的促进作用。本论文对HCF-1和NSPc-1调控细胞周期的分子机制进行了研究。采取RNA干扰的方法发现，敲低HCF-1后Cdc42的表达水平降低，但是敲低Cdc42的表达水平对HCF-1基本没有影响。这说明HCF-1具有促进Cdc42基因表达的作用。染色质免疫沉淀和DNA亲和层析实验都发现，HCF-1可以特异性结合于Cdc42基因启动子区(-1060～-644)区段。上述结果说明，HCF-1在转录水平调控Cdc42基因的表达。HCF-1对Cdc42的表达调控作用也是与细胞周期的进程紧密相关的。HCF-1在S期达到表达高峰，Cdc42的表达高峰稍迟于HCF-1。并且，HCF-1在S期结合于Cdc42基因启动子的蛋白量也相应高于G1期和M期。这说明，HCF-1在S期的高表达造成HCF-1在Cdc42基因启动子区的结合量增加，从而促进了Cdc42的表达，因此Cdc42的表达高峰稍迟于HCF-1。HCF-1对细胞G1-S期进程和有丝分裂具有重要的调控作用。HCF-1表达敲低后，细胞增殖减慢，BrdU掺入率降低，G1-S期进程出现阻滞，cylin A基因的表达降低，有丝分裂中期染色体在赤道板出现排列异常和多核现象。Cdc42表达敲低后细胞也出现了类似的表型。过表达Cdc42基因的组成活性突变体Cdc42F28L可以在一定程度上挽救HCF-1表达敲低导致的细胞G1-S期进程阻滞、cyclin A基因表达降低和多核现象发生的比率。上述结果说明，HCF-1对G1-S期进程和有丝分裂的调控作用是通过其下游靶基因Cdc42实现的。实验中还检测了NSPc-1稳定过表达和稳定表达敲低的细胞系中7种主要CDKI分子的表达变化时发现，NSPc-1稳定过表达细胞中p21基因的表达水平出现降低，NSPc-1稳定表达敲低的细胞中p21基因的表达升高。这提示p21基因可能是NSPc-1的调控靶基因。实验中还发现，NSPc-1结合于p21基因启动子区(-1357～-1063)区段，并依赖于其中-1203位的维甲酸受体反应元件(RARE)。p21基因具有抑制G1-S期进程的作用，因此这就可以初步解释NSPc-1促进细胞G1-S期进程的分子机制。综上所述，宿主细胞因子HCF-1转录水平调控促进Cdc42基因的表达，并且HCF-1对G1-S期进程和有丝分裂的调控作用是通过其靶基因Cdc42介导的。多梳蛋白NSPc-1通过抑制p21基因的转录对G1-S期进程发挥的调控作用，并且NSPc-1对p21基因启动子区的调控作用依赖于上游—1203位的维甲酸受体反应元件(RARE)。更多还原

【Abstract】 Host cell factor 1 (HCF-1) as a requisite component of chromatin related complexduring cell replication pocesses the function on cell-cycle progression. In mammalian cellswith HCF-1 knockdown, most cells are arrested in G1 and fail to enter S phase. Moreover,normal mitosis is also affected with more or less extent of multi-nucleation in different celllines. Cdc42 as one of the most characterized members of Rho GTPases has been greatlyproved to have the similar abilities to regulate cell cycle progression. NSPc-1 was firstidentified in 2000 as the new PcG member, which is ubiquitously expressed in all kinds ofhuman adult tissues. Our published data have identified that as a typical PcG member,NSPc-1 has transcriptional repression activity and could promote G1-S progression of cellcycle. This PhD dissertation is mainly focused on the studies of molecular mechanisms ofHCF-1 and NSPc-1 on cell cycle regulation.RNA interference assays found that Cdc42 expression level is decreased inaccompany with loss of HCF-1 function, but HCF-1 unaffected after Cdc42 RNAi. Theresult demonstrates that HCF-1 could promote the expression of Cdc42.Sequentially Chromatin Immunoprecipitaion assay and DNA pulldown assays bothclarify the exact binding of HCF-1 on Cdc42 promoter (-1060～-644). Taken together,HCF-1 transcriptionally regulates Cdc42 expression.The effect of HCF-1 on regulating Cdc42 expression is associated with cell cycleprogression. The highest level of HCF-1 is appeared in synchronized population at S phase,while Cdc42’s is sequentially appeared with several hours later. The binding ratio ofHCF-1 on Cdc42 promoter at S phase is consequently higher than G1 phase and M phasepopulation. It is consequently concluded that the higher expression level of HCF-1 at Sphase leads to more HCF-1 binding on Cdc42 promoter, which then promotes theexpression of Cdc42. Therefore, the expression peak of Cdc42 is appeared after HCF-1with a little delay.HCF-1 has important function on G1-S progression and mitosis. After knockdown of HCF-1, cell proliferation is delayed, cell cycle is arrested at G1 phase with decreased BrdUincorporation and lower expression of cyclin A, chromsome misalignment andmulti-nucleation appear. Cdc42 as the target gene regulated by HCF-1 also possess thesimilar abnormal phenotype after expression knockdown. Over-expression of constituentactive mutant Cdc42F28L could rescue the abnormal phenotype after HCF-1 knockdown,such as lowered BrdU incorporation, decreased cyclin A expression level and BrdU lowerincoporation. Taken together, the function of HCF-1 on G1-S progression and mitosis isachieved through its transcriptional target Cdc42.Screening of 7 CDKIs within NSPc1 stable overexpressed and NSPc1 stableknockdowned cell lines found that only the expression level of p21 was repressed whenNSPcl overexpressed; Correspondently, p21 was upregulated in NSPc1 stablyknockdowned cell lines. This infers that p21 may be the target gene regulated by NSPc1.Moreover, Chromatin Immunoprecipitaion assay and DNA pulldown assays wereused to find that NSPc1 could bind the region (-1357～-1063) of p21 promoter, and itsbinding activity is dependent on retinoic acid receptor response element (RARE) located at-1203 within the above region, p21 gene has been identified to have the inhibitory effect onG1-S progression, therefore it is believable that the effect of NSPc1 on G1-S progression isdependent on its transcriptional regulation on p21.In summary, HCF-1 on the transcriptionl level regulates the expression of Cdc42,which mediate its effect on G1-S progression and mitosis. NSPc-1 transcriptionally inhibitsp21 gene expression, which mediates its effect on G1-S progression. The effect ofNSPc-1 on regulating p21 promoter is dependent on the retinoic acid receptor responseelement (RARE) located at-1203.更多还原