Molecular Mechanisms of Transcriptional Specificity in Cell Cycle and Oncogenic Pathways

 Joseph Nevins^1,2,4, Erich Huang^1,4, Esther Black^1,4, Holly Dressman^1,4 and Mike West^3,4

¹Department of Molecular Genetics and Microbiology, Duke University Medical Center; ²Howard Hughes Medical Institute; ³Institute of Statistics and Decision Sciences, Duke University; ⁴Computational and Applied Genomics Program, Duke Institute for Genome Sciences and Policy

A central component of the control of normal cellular proliferation is the action of Rb in the control of the E2F-family of transcription factors. E2Fs activate a large array of genes that encode proteins important for DNA replication and cell cycle progression but also the p53 response pathway and additional components of the cell death machinery, thus linking cell proliferation with cell fate determination.  These studies suggest distinct roles for individual E2F proteins, likely coupled with specificity of transcription activation. Additional work has linked the action of the Myc oncoprotein with the Rb/E2F pathway since Myc induces E2F accumulation and the ability of Myc to induce S phase and apoptosis is dependent on these E2F activities. Taken together, these observations suggest a complex interplay of gene regulatory pathways that control both cell proliferation and the ultimate decisions of cell death. We have explored the specificity of E2F transcription control using biochemical approaches, primarily through the analysis of specific chromatin interactions of key regulatory proteins. Our previous work has identified TFE3 as a transcription partner for the E2F3 protein in the control of the DNA polymerase a p68 gene.  Additional work has identified YY1 as a partner for E2F2 and E2F3.  We have extended these studies through the analysis of promoter sequences that share common elements and promoter architecture with the previously defined regulated promoters. This has revealed several additional genes that share the same structure and that are also regulated by the combined action of TFE3 and E2F3.  Other genes such as thymidine kinase (TK-1) are also regulated by E2F3 but independent of TFE3.  Yet other E2F target genes exhibit distinct specificity in the interaction with E2F proteins that includes a role for E2F1 but not E2F3 and examples where both E2F1 and E2F3 are seen to interact. Finally, the combinatorial nature of this control is emphasized by the role of TFE3 in the transcription of other groups of genes that is independent of the E2Fs.  Further studies in progress are making use of large scale chromatin immunoprecipitations coupled with DNA microarrays to assess a more global context of the combinatorial interactions involving E2F proteins.

We have also focused on the broader questions of global gene control in these pathways using genome-scale analysis of gene expression. Large scale analysis of gene expression derived from DNA microarray studies has the potential for adding enormous information to the analysis of biological phenotypes. We have applied strategies to identify gene expression phenotypes of oncogenic signaling pathways including Ras, Myc, and Rb-E2F pathways. Our analyses identify patterns of gene expression that define the function of these pathways and that can accurately predict the activity of these pathways.  Importantly, these analyses have the capacity to dissect related but distinct aspects of E2F function, identifying gene expression patterns that can distinguish the action of the E2F1, E2F2, and E2F3 activator proteins.  In addition, the development of these gene expression profiles has shown an ability to predict the deregulation of these oncogenic signaling pathways in the context of mouse tumor models. We believe that these gene expression phenotypes have the potential to characterize the complex genetic alterations that typify the neoplastic state in a way that truly reflects the complexity of the regulatory pathways that are affected.