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Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line

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 pancr...

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Autores principales: Lizio, Marina, Ishizu, Yuri, Itoh, Masayoshi, Lassmann, Timo, Hasegawa, Akira, Kubosaki, Atsutaka, Severin, Jessica, Kawaji, Hideya, Nakamura, Yukio, Suzuki, Harukazu, Hayashizaki, Yoshihide, Carninci, Piero, Forrest, Alistair R. R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650373/
https://www.ncbi.nlm.nih.gov/pubmed/26635867
http://dx.doi.org/10.3389/fgene.2015.00331
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author Lizio, Marina
Ishizu, Yuri
Itoh, Masayoshi
Lassmann, Timo
Hasegawa, Akira
Kubosaki, Atsutaka
Severin, Jessica
Kawaji, Hideya
Nakamura, Yukio
Suzuki, Harukazu
Hayashizaki, Yoshihide
Carninci, Piero
Forrest, Alistair R. R.
author_facet Lizio, Marina
Ishizu, Yuri
Itoh, Masayoshi
Lassmann, Timo
Hasegawa, Akira
Kubosaki, Atsutaka
Severin, Jessica
Kawaji, Hideya
Nakamura, Yukio
Suzuki, Harukazu
Hayashizaki, Yoshihide
Carninci, Piero
Forrest, Alistair R. R.
author_sort Lizio, Marina
collection PubMed
description 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.
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spelling pubmed-46503732015-12-03 Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line Lizio, Marina Ishizu, Yuri Itoh, Masayoshi Lassmann, Timo Hasegawa, Akira Kubosaki, Atsutaka Severin, Jessica Kawaji, Hideya Nakamura, Yukio Suzuki, Harukazu Hayashizaki, Yoshihide Carninci, Piero Forrest, Alistair R. R. Front Genet Genetics 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. Frontiers Media S.A. 2015-11-18 /pmc/articles/PMC4650373/ /pubmed/26635867 http://dx.doi.org/10.3389/fgene.2015.00331 Text en Copyright © 2015 Lizio, Ishizu, Itoh, Lassmann, Hasegawa, Kubosaki, Severin, Kawaji, Nakamura, FANTOM consortium, Suzuki, Hayashizaki, Carninci and Forrest. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Lizio, Marina
Ishizu, Yuri
Itoh, Masayoshi
Lassmann, Timo
Hasegawa, Akira
Kubosaki, Atsutaka
Severin, Jessica
Kawaji, Hideya
Nakamura, Yukio
Suzuki, Harukazu
Hayashizaki, Yoshihide
Carninci, Piero
Forrest, Alistair R. R.
Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line
title Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line
title_full Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line
title_fullStr Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line
title_full_unstemmed Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line
title_short Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line
title_sort mapping mammalian cell-type-specific transcriptional regulatory networks using kd-cage and chip-seq data in the tc-yik cell line
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650373/
https://www.ncbi.nlm.nih.gov/pubmed/26635867
http://dx.doi.org/10.3389/fgene.2015.00331
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