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An embryonic stem cell-based system for rapid analysis of transcriptional enhancers.

ABSTRACT: With the growing use of genome-wide screens for cis-regulatory elements, there is a pressing need for platforms that enable fast and cost-effective experimental validation of identified hits in relevant developmental and tissue contexts. Here, we describe a murine embryonic stem cell (ESC)-based system that facilitates rapid analysis of putative transcriptional enhancers. Candidate enhancers are targeted with high efficiency to a defined genomic locus via recombinase-mediated cassette exchange. Targeted ESCs are subsequently differentiated in vitro into desired cell types, where enhancer activity is monitored by reporter gene expression. As a proof of principle, we analyzed a previously characterized, Sonic hedgehog (Shh)-dependent, V3 interneuron progenitor (pV3)-specific enhancer for the Nkx2.2 gene, and observed highly specific enhancer activity. Given the broad potential of ESCs to generate a spectrum of cell types, this system can serve as an effective platform for the characterization of gene regulatory networks controlling cell fate specification and cell function.

SUBMITTER: Tsanov KM

PROVIDER: S-EPMC3334447 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other

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An embryonic stem cell-based system for rapid analysis of transcriptional enhancers.

Tsanov Kaloyan M KM Nishi Yuichi Y Peterson Kevin A KA Liu Jing J Baetscher Manfred M McMahon Andrew P AP

Genesis (New York, N.Y. : 2000) 20120117 5

With the growing use of genome-wide screens for cis-regulatory elements, there is a pressing need for platforms that enable fast and cost-effective experimental validation of identified hits in relevant developmental and tissue contexts. Here, we describe a murine embryonic stem cell (ESC)-based system that facilitates rapid analysis of putative transcriptional enhancers. Candidate enhancers are targeted with high efficiency to a defined genomic locus via recombinase-mediated cassette exchange. ...[more]

PMID: 22083581

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Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the method of choice to study function of transcription factors in cell fate determination. This technique faces two major obstacles when applied to developmental studies: the availability of ChIP grade antibodies and access to sufficient quantities of the cells of interest. We present a robust method for the genome-wide analysis of transcription factor binding during development in highly homogenous cells in defined developmental states. It combines efficient embryonic stem cell (ESC) differentiation protocols with an inducible system of tagged transcription factors to enable affinity based assays such as ChIP-seq during lineage specific development. To validate the system, we compared the activity and genomic binding of native and V5-tagged Olig2 in motor neuron progenitors and Flag-tagged and V5-tagged Hoxc9 in motor neurons. We find that tagging transcription factors and expressing them alongside their endogenous counterparts does not alter their function or genomic association. The technology presented here can be applied to known as well as novel DNA-binding proteins. In combination with a suitable ESC differentiation paradigm it can be applied to determine how lineage-specific transcriptional networks are established and regulated. The aim of this study is to validate a platform for analysis of transcription factor binding that combines directed differentiation of ES cells with an inducible system of tagged transcription factors. Here, we perform ChIP-seq analysis to compare binding profiles of Olig2 as found using a native antibody and anti-V5 against the epitope tag. We also compare the ChIP-seq profiles of Hoxc9 as found using two independent epitope tags (V5 and FLAG). In all, there are 6 Illumina sequence datasets in this submission, including one replicate for each of native Olig2, iOlig2-V5 and FLAG-iHoxc9, two replicates of iHoxc9-V5, and a pseudo-ChIP control using anti-V5 in a non-induced iOlig2 cell-line. The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog.

Project description:Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and approximately 350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs.

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An embryonic stem cell-based system for rapid analysis of transcriptional enhancers.

Publications

An embryonic stem cell-based system for rapid analysis of transcriptional enhancers.

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