3.1. Differentially expressed genes and affected processes
To begin analysis DE genes in direct reprogramming of mouse fibroblast to iNS cells were detected. Collectively 2167 DE genes include 1020 up and 1147 down regulated genes (File S1). Cluster with a highest enrichment score in functional clustering analysis of DE genes contain signal term as a top GO term (552 DE genes). Cluster that accommodate terms related to nervous system assign enrichment score 3.04 to itself and regulation of nervous system development was the top term of this cluster with 29 DE genes and p value equal to 0.0018 (File S2).
3.2. TFs involved in regulation of DE genes in the direct conversion of mouse ...view middle of the document...
Third complex of protein-protein interaction had the lowest score (2) and contain only two TFs (TET1 and SIN3A) with two interactions. Collectively 14 TFs out of 46 TFs identified to involve in complexes during direct reprogramming of mouse fibroblast to iNS cells.
3.4. Central TFs in gene regulatory network during direct conversion of mouse fibroblast to iNS cells
To find central TFs, we integrate protein-DNA interactions, protein-protein interactions and expression data to build a gene regulatory network (1857 nodes and 11054 genes) (Figure.2). Based on eccentricity analysis NANOG, POU5F1, SOX2, MYC, TCF3, SALL4, and NR0B1 have the same value and all of them ranked as top central genes. Closeness parameter ranked SUZ12, POU5F1, NANOG, PPARG, TCF3, SOX2, MTF2, MYC, and SMARCA4 as ten top central regulators in gene regulatory network. Centrality analysis according to stress parameters identified NANOG, POU5F1, SOX2, MTF2, MYC, PPARG, SMARCA4, EZH2, and TCF3 as ten TFs with highest values in gene regulatory network. Top ten TFs revealed by betweenness are PPARG, MTF2, NANOG, SMARCA4, MYC, POU5F1, SOX2, TCF3, and EZH2. Most of 46 TFs have same value based on centroid analysis (Table.1). Finally, based on six centrality parameter NANOG, POU5F1, PPARG, MTF2, SOX2, MYC, SUZ12, TCF3, SMARCA4, EZH2, JARID2, TET1, SALL4, OLIG2, and TRIM28 introduced as top 15 central TFs in constructed gene regulatory network during direct reprogramming of adult fibroblast to iNS cells (Table.1).
3.5. Most important TFs in expression based modules
To find most important modules and TFs based on expression JActiveModule was used.TFs includes CEBPB, STAT4, SUZ12, RAD21, TCF3, GATA3, TFCP2L1, WT1, OLIG2, EZH2, JARID2, NANOG, SALL4, SOX2, NR0B1, MYB, ZIC3, and POU5F1 are present in 5 top active modules during conversion of mouse fibroblast to iNS cells (Figure.2). Top modules ontology revealed neural tube formation as most important related GO term based on the number of involved DE genes; Adm, Celsr1, Cthrc1, Sall4, Sox11, Zic2, and Zic5 were found in third and fourth active modules. Main regulators of these 7 genes are EZH2, SUZ12, and NANOG (6, 6 and 5 targets respectively), such as EZH2 bind to promoters of Adm, Celsr1, Sall4, Sox11, Zic2, and Zic5 genes.
3.6. Nervous system development genes in gene regulatory network and their regulators in direct reprogramming of adult fibroblast to iNS cells.
Based on gene regulatory network ontology we identified nervous system development term with 236 DE genes (p value = 4.15*10-19). We analyzed this list to find main regulators in iNS cells converted from adult fibroblast. Interestingly 14 out of 46 DE TFs include CEBPB, PPARG, RUNX1, FOXP2, NR3C1, PRDM16, SMARCA4, GATA3, OLIG2, EZH2, MYC, SALL4, SOX2, and ZIC3 identified to be members of nervous system development term in the gene regulatory network ontology. Main regulators of this list are SUZ12, MTF2, POU5F1, NANOG, EZH2, TCF3, SOX2, JARID2, SMARCA4, and MYC that ranked...