290 Current Genomics, 2010, Vol. 11, No. 4 To gain further insight into potential biological pathways altered in our transgenic cells relative to the wildtype cells, we carried out category/pathway analysis. More specifically, we performed Gene Set Analysis (GSA) [17]. In contrast to SGA, GSA assesses statistical significance of a gene set/pathway as a whole. This analysis allows for identifica- tion of gene sets/pathways with genes that show coordinated changes in expression, even if the changes are modest. SGA and GSA are two complementary approaches. SGA identi- fies any transcript that meets a chosen filtering criteria such as a specific p-value cutoff independent of a pathway con- tent, whereas GSA identifies gene sets/pathways with genes that share a common functional theme. We examined gene sets organized by Gene Ontology Biological Process (BP). Table 2 lists gene sets with p<0.001. GSA reveals suppres- sion of gene sets involved in embryonic eye morphogenesis, gut development, water transport, fluid transport and regula- tion of adenylate cyclase activity in the transgenic cells, after serum stimulation. Components of these gene sets may be direct targets of transcriptional repression by CHF1/Hey2. Analysis of gene sets that are increased in transgenic relative to WT cells also revealed a number of interesting Yu et al. pathways. In the absence of serum, gene sets involved in regulation of transcription factor activity, protein export from the nucleus, steroid hormone receptor signaling and regulation of protein phosphatase activity are increased. Similar gene sets involved in protein dephosphorylation and dephosphorylation are increased both in the absence and presence of serum. The individual components of these re- lated gene sets overlap significantly, and their significance scores are largely driven by the same genes. In the presence of serum, multiple gene sets involved in autophagy, apopto- sis, mitochondrial biogenesis, cellular component disassem- bly and viral reproduction demonstrate increased expression. Although CHF1/Hey2 is considered to be a transcriptional repressor, activation of multiple pathways may reflect either positive activation by CHF1/Hey2 or repression of negative regulatory factors for these pathways. Analysis of individual components from various BP gene sets suppressed by CHF1/Hey2 reveals multiple potential targets that may play a role in hypertrophy. These include genes involved in water transport (GO:0006833) such as aquaporins 2, 3, 5, 6, 7, 8, 9 and 11, suggesting that CHF1/ Hey2 globally suppresses water transport and that water Table 2. Gene Ontology Biological Process (BP) Gene Set Changes in CHF1/Hey2 Transgenic Myocytes vs Wild Type Myocytes before and/or after Serum Stimulation Tg vs. WT, No Serum Tg vs. WT, Serum GO:0006611 GO:0030518 GO:0016311 GO:0048048 GO:0048565 GO:0006833 GO:0042044 GO:0016236 GO:0000045 GO:0022411 GO:0022415 GO:0030262 GO ID Biological Process GO:0006470 protein amino acid dephosphorylation GO:0051090 regulation of transcription factor activity GO:0043666 regulation of phosphoprotein phosphatase activity protein export from nucleus steroid hormone receptor signaling pathway dephosphorylation embryonic eye morphogenesis gut development water transport fluid transport macroautophagy autophagosome formation cellular component disassembly viral reproductive process apoptotic nuclear changes GO:0045761 regulation of adenylate cyclase activity GO:0006309 DNA fragmentation during apoptosis GO:0006921 cell structure disassembly during apoptosis GO:0007005 mitochondrion organization and biogenesis 137 Size 132 155 77 20 75 10 10 33 16 18 55 16 13 17 22 22 59 25 p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.126 0.167 0.038 0.064 0.019 0.11 0.074 0.337 0.214 0.09 0.214 0.213 0.483 FDR <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Score 0.5438348 0.4408056 0.806123 0.3664094 1.2864495 0.4924526 0.7716088 -0.3379658 0.7736608 -0.1805751 0.7716088 -0.8349875 0.7716088 -0.6696553 0.7716088 -0.3603997 1.0068723 0.3604726 0.9947778 0.5173092 0.8456392 -0.1261624 0.792464 -0.2620884 0.9947778 0.4035484 0.792464 -0.2620884 1.074234 0.2154094 1.1064093 0.0040825 p-value <0.001 0.465 0.147 0.159 0.081 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 FDR <0.001 Score 0.3885387 1.0867179 0.013523 0.9996068 0.2632729 0.9996068 0.1481744 0.9127665 0.5832501 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.3715223 -0.6525282 -0.6338953 -1.2825472 -1.1006198 -0.7334642 1.04785 1.223408 0.9596088 0.6978587 0.6073059 0.6978587 0.4824776 0.6125828