Time Course of Gene Expression in Rat-1 Fibroblasts Following Alpha-1a Adrenergic Receptor Stimulation with Phenylephrine.

In mammals, increasing evidence supports the generality of cotranscriptional gene regulation and the idea that genetic control often occurs subsequent to RNA polymerase II (Pol II) recruitment. In our recent experiments, we have used Pol II Chromatin Immunoprecipitation (ChIP) to investigate relationships between the mechanistic events that control acute stress response (ASR) gene activation following stimulation of the alpha1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate the temporal accuracy of our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Initial temporal microarray analysis of gene activation following agonist addition identified genes that were upregulated and provided a temporal profile of changes in polyadenylated mRNA levels. Combined with PCR analysis of pre-mRNA, total RNA, polyadenylated RNA and the Pol II ChIP data, we demonstrate complex multilevel gene regulation including a diverse set of cotranscriptional mechanisms. Given our data, we propose that analysis of a genes regulation must begin with global assessement of changes in transcriptional behavior to identify events that are actually rate limiting if reductionist mechanistic dissection is to distinguish events that are regulated from the larger set of mechanisms available to most genes. This microarray analysis reports on the temporal changes in polyadenylated mRNA levels in proliferating rat-1 fibroblasts following stimulation of the alpha-1a Adrenergic Receptor with filter sterilized 10 uM phenylephrine (PE). Cells were treated and harvested at 70 to 80% confluence. Rat-1 cells under these conditions are still mid-log phase and continue to proliferate for at least another replicative cycle. Message levels in untreated cells were determined for 3 biological replicates, but all other time points are single replicates as sufficiently detailed dissection made replicates expensive and unnecessary, given that clear trending behavior in the expression profiles demonstrates profile accuracy.