Elucidating the Regulation of Delayed-Early Genes by Sustained MAP Kinase Pathway Activation

RAS and its downstream effector, BRAF, are commonly mutated proto-oncogenes in many types of human cancer. Mutationally activated RAS or BRAF signal through the MEK→ERK MAP kinase pathway to regulate key cancer cell hallmarks such as progress through the cell cycle and reduced cell death. We classified positively RAS/RAF-regulated genes into two lists, immediate-early and delayed-early response genes (IEG and DEG). Canonical targets of sustained RAS/RAF activation that are also resistant to protein synthesis inhibitor, cycloheximide, are IEG. Expression of genes that require sustained, high-level activation of MAPK signaling mediated by oncogene activation and are sensitive to cycloheximide we classified as DEG. We have identified over 300 genes that fit the DEG classification using RNA-sequencing. However, to date, the regulatory mechanisms for this class of genes remain obscure. We use integrin b3 (ITGB3) as model to test our hypothesis that there is a different mechanism of regulation for DEG compared to canonical regulation of IEG. To determine if there is an underlying regulatory logic to the observed effects of activated RAS/RAF delayed-early genes we have linked altered expression of DEG with induced changes in chromatin structure using high-throughput sequencing, CRISPR activation and interference, and chromatin immunoprecipitation of active transcription marks.