title: Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line.
abstract: 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.
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan.
authors.forename: Marina
authors.initials: M
authors.lastname: Lizio
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Ishizu
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; RIKEN Preventive Medicine and Diagnosis Innovation Program Yokohama, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Itoh
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; Telethon Kids Institute, The University of Western Australia Subiaco, WA, Australia.
authors.forename: Timo
authors.initials: T
authors.lastname: Lassmann
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hasegawa
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan.
authors.forename: Atsutaka
authors.initials: A
authors.lastname: Kubosaki
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan.
authors.forename: Jessica
authors.initials: J
authors.lastname: Severin
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; RIKEN Preventive Medicine and Diagnosis Innovation Program Yokohama, Japan.
authors.forename: Hideya
authors.initials: H
authors.lastname: Kawaji
authors.affiliation: Cell Engineering Division, RIKEN BioResource Center Ibaraki, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan.
authors.forename: Harukazu
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; RIKEN Preventive Medicine and Diagnosis Innovation Program Yokohama, Japan.
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan.
authors.forename: Piero
authors.initials: P
authors.lastname: Carninci
authors.affiliation: RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; QEII Medical Centre and Centre for Medical Research, Harry Perkins Institute of Medical Research, The University of Western Australia Nedlands, WA, Australia.
authors.forename: Alistair R R
authors.initials: AR
authors.lastname: Forrest
keywords: CAGE; ChIP-seq; FANTOM5; pancreas; perturbation; transcriptional regulatory network