EZH2 isoforms can regulate gene expression genome-wide through defined subnetworks. To test if EZH2β- or EZH2α-specific genes form interdependent, unique subnetworks, Affymetrix data generated in A was parsed for targets uniquely and significantly repressed (P <0.05, log2 fold change >−2) by EZH2β, EZH2α or both compared with empty vector. No change is defined as P >0.05 and log2 fold change between 1.5 and −1.5. Subnetworks were reconstructed using an IPA-propriety algorithm. While a multitude of subnetworks were generated, a high scoring representative example network for each condition is shown. (A) IPA-assisted subnetwork analysis indicates that EZH2β participates in the regulation of genes involved in several ontological categories, including cellular function and maintenance as well as hematological system function. Overexpression of EZH2β results in significant repression whereas EZH2α overexpression fails to produce the same repressive effects in this particular subnetwork. (B) Similar subnetwork analysis of expression data for enrichments of biological function mediated by EZH2α indicates that this protein regulates targets associated with proliferative responses and cell cycle regulation. (C) Subnetwork analysis of expression data for enrichments of biological function mediated by both EZH2β and EZH2α indicates enrichment of targets involved in cell death and survival and cell signaling. Combined, these data demonstrate that that novel EZH2 isoforms can regulate gene expression genome-wide through unique and shared targets that are interconnected to form defined subnetworks. Note that, although both EZH2 isoforms can often regulate different genes represented by the examples (A, B and C), the subnetworks formed by these genes are ontologically known to participate in similar processes (B). This knowledge not only is congruent with the ability of both EZH2 proteins to regulate cell growth as revealed by our cell biology assays (Figure 4E), but also expands the potential functional association of these isoforms. EZH2: enhancer of zeste homologue 2.