Co-expression and functional assay of human Rb and E2F1 proteins in yeast

[摘要] The mammalian cell cycle is composed of a myriad interactions occurring in a definedsequence dictated by the flow of entropy. The decision to study the cell cycle requiresentry into a world where events take place, not because they want to, but because they haveto. Cells do not 'decide' to perform certain actions, but are driven by the laws of nature, thesame laws that are responsible for the existence of the universe. Study of this maelstrom ofreactions is truly analogous to opening Pandora's proverbial box. A peek inside and all theinner workings of the cell start to spill out. Unfortunately, this is where things start to getcomplicated.Serendipitously, simpler alternatives are available. The yeast cell cycle is remarkablysimilar to that of higher animals. The Rb and E2F 1 proteins are integral components of themammalian cell cycle, and as such, they are found in aberrant forms in numerousmalignancies. This necessitates an adequate means for the determination of the cellularstatus of these proteins, as prognosis and diagnosis of several neoplastic disorders aredependentthereon.This study aimed to develop a yeast-based strategy for functional analysis of the humantumour suppressor protein Rb. This goal was impeded by one factor; the yeast cell cycle istoo similar to that of humans. Several yeast strains, derived from W303-lA, wereconstructed that each contain a reporter gene -the lacZ gene of E. coli- regulated by E2Frecognition elements introduced within the upstream CyC1 promoter. The theory was thatin the absence of Rb, ectopically expressed human E2F 1 protein would be able to bind theRE and activate transcription of the reporter gene, ultimately resulting in a readilyobservable product. In the presence of functional Rb, E2Fl would be bound by the tumoursuppressor protein, and thus be incapable of activating the reporter gene. This wouldprovide an assay of Rb status, based not on tedious sequencing analysis of the geneticmaterial, but on the actual functional activity of the protein.When dealing with Mother Nature, though, we are oftentimes reminded that she hasthought of everything. S. cerevisiae contains an E2F-like activity, capable of binding theexact RE introduced into the reporter gene promoter. This was confirmed by experimentsin this study, where the reporter gene was activated in the absence of ectopically expressedE2F1 protein. The endogenous yeast E2F-like protein is thus able to activate transcriptionof the reporter gene, negating the effect that would be observed by ectopic expression ofhuman E2F 1, and thus, all Rb-expression was performed in the absence of eo-expressedhuman E2F 1. Since it is impossible to distinguish between the yeast and human E2Factivities, it is impossible to create a functional assay for Rb activity in the W303-1Aderivedchimeras constructed.As mentioned previously, it could be possible to overcome this problem by knocking outthe yeast-borne E2F activity, but this approach is restricted by two barriers. Firstly, theyeast equivalent of the E2F-family is yet to be cloned. This problem can be approachedwith a transposon-based strategy. The endogenous E2F activity is capable of activatingtranscription of the lacZ reporter gene, and in so doing, provides a convenient assay forYE2F integrity. Through the use of a plasmid containing an inducible transposase it shouldbe possible to disrupt the YE2F -encoding gene through integration of a transposon. Thiswould be accompanied by an inability of the yeast to activate the reporter gene. Thetransposon, containing flanking genomic DNA, could then be retrieved and provide thebasis for cloning the gene coding for YE2F. Since the reporter gene is specific for E2F-likebinding, retrieval of non-specific factors should be negated.A second problem is that, since the E2F proteins play an important role in the progressionof the cell cycle in higher animals, it is possible that the yeast would not survive knockingout its homologue. Relegation of YE2F to the role of bystander could possibly wreakhavoc with the delicate mechanism that is the cell cycle. Still, it would be interesting tofurther pursue this idea.Ectopic expression of human Rb in the various strains used in this study provided someinteresting results. Transformation of pAWl, followed by galactose-based induction of Rbexpression resulted in observable differences in growth characteristics of certain strains.Those most affected were W-lf and W-1r, which each contain a single repeat of the E2FRE within the upstream promoter of the reporter gene, albeit in forward and reverseorientations, respectively. The effect was particularly evident in W-1r, where expression ofRb resulted in complete cessation of growth, probably due to binding of yeast E2F-likeactivity. These results could not, however, be reconciled with those obtained from cellcycle analysis with the aid of flow cytometry. These experiments did not show anysignificant effect of Rb expression on the cell cycle of the examined strains. This ispossibly due to an insufficient period of observation, but this could, unfortunately, neitherbe confirmed nor dismissed.From the results obtained in this study it appears that construction of an apposite reportersystem for the functional assay of Rb is perhaps more tricky than would be expected. Theinterference of endogenous proteins is a cause for concern in a development strategy suchas this, and serves as a caveat for future studies in this field.

[发布机构] University of the Free State