Identification of transcriptional regulatory cascades in retinoic acid-induced growth arrest of HepG2 cells.

All-trans retinoic acid (ATRA) is a potent inducer of cell differentiation and growth arrest. Here, we investigated ATRA-induced regulatory cascades associated with growth arrest of the human hepatoma cell line HepG2. ATRA induced >2-fold changes in the expression of 402 genes including 55 linked to cell-cycle regulation, cell growth or apoptosis during 48 h treatment. Computational search predicted that 250 transcriptional regulatory factors (TRFs) could recognize the proximal upstream regions of any of the 55 genes. Expression of 61 TRF genes was significantly changed during ATRA incubation, providing many potential regulatory edges. We focused on six TRFs that could regulate many of the 55 genes and found a total of 160 potential edges in which the expression of each of the genes was changed later than the expression change of the corresponding regulator. RNAi knockdown of the selected TRFs caused perturbation of the respective potential targets. The genes showed an opposite regulation pattern by ATRA and specific siRNA treatments were selected as strong candidates for direct TRF targets. Finally, 36 transcriptional regulatory edges were validated by chromatin immunoprecipitation. These analyses enabled us to depict a part of the transcriptional regulatory cascades closely linked to ATRA-induced cell growth arrest.