HCF-1 And NSPc-1 Regulation Of Cell Cycle Molecular Mechanisms

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.