Cell Cycle Regulation
Most assays which measure cell cycle-regulated transcription do so by monitoring either promoter activity or mRNA accumulation on a population basis. For example, we have clearly shown that cad (a Myc target gene) and dhfr (an E2F target gene) mRNAs increase in abundance at the G1/S phase boundary when mRNA is prepared from dishes of cultured cells and that the cad and dhfr promoters display increased transcriptional activity at the G1/S phase boundary when monitored using a promoter-reporter assay (Miltenberger et al., 95; Slansky et al., 93). Taken together, these measurements have suggested a 10-20 fold increase in promoter activity as cells progresss from G0 to S phase. However, these studies do not reveal whether a 10-20 fold increase is occuring in every cell on the dish or whether the increase reflects an average value, with some cells displaying a higher fold increase and some cells showing little or no increase in transcriptional activity. Our recent studies have suggested that for the cad (Boyd, MCB 99) and dhfr (Wells et al., 2000) promoters, several different transcription complexes have the potential to form, even in a synchronized cell population. These results raise the possibilities that either the different transcription complexes are functionally redundant or that different cells in a clonal population are responding differently to environmental stimuli. We are currently using two different experimental approaches to distinguish these possibilitiese. First, we are analyzing mRNA accumulation on a single cell basis using in situ hybridizations and second, we are separating functional from nonfunctional transcription complexes (using cell sorting) and identifying the specific transcription factors which compose each type of complex.