TY - JOUR AU - Lizio, Marina AU - Ishizu, Yuri AU - Itoh, Masayoshi AU - Lassmann, Timo AU - Hasegawa, Akira AU - Kubosaki, Atsutaka AU - Severin, Jessica AU - Kawaji, Hideya AU - Nakamura, Yukio AU - , FANTOM consortium AU - Suzuki, Harukazu AU - Hayashizaki, Yoshihide AU - Carninci, Piero AU - Forrest, Alistair R. R. PY - 2015 M3 - Original Research TI - Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line JO - Frontiers in Genetics UR - https://www.frontiersin.org/articles/10.3389/fgene.2015.00331 VL - 6 SN - 1664-8021 N2 - Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP-seq datasets. (1) A large fraction of binding sites are at distal enhancer sites and cannot be directly associated to their targets, without chromatin conformation data. (2) Many peaks may be non-functional: even when there is a peak at a promoter, the expression of the gene may not be affected in the matching perturbation experiment. ER -