Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

CTCF functions as a chromatin insulator in the HoxA cluster during neurogenesis [RNA-Seq]

ABSTRACT: In this experiment, we sought to analyze how the transcriptome of WT, Δ5|6, and Δ5|6:7|9 cells vary during differentiation of ESCs into cervical motor neurons 3 lines (WT, Δ5|6, Δ5|6:7|9)

ORGANISM(S): Mus musculus

SUBMITTER: Danny Reinberg

PROVIDER: E-GEOD-60226 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.

Narendra Varun V Rocha Pedro P PP An Disi D Raviram Ramya R Skok Jane A JA Mazzoni Esteban O EO Reinberg Danny D

Science (New York, N.Y.) 20150201 6225

Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into ...[more]

PMID: 25722416

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Project description:Transcriptional programming of cell identity promises to open up new frontiers in regenerative medicine by enabling the efficient production of clinically relevant cell types. We examine if such cellular programming is accomplished by transcription factors that each have an independent and additive effect on cellular identity, or if programming factors synergize to produce an effect that is not independently obtainable. The combinations of Ngn2-Isl1-Lhx3 and Ngn2-Isl1-Phox2a transcription factors program embryonic stem cells to express a spinal or cranial motor neuron identity respectively. The two alternate expression programs are determined by recruitment of Isl1/Lhx3 and Isl1/Phox2a pairs to distinct genomic locations characterized by two alternative dimeric homeobox motifs. These results suggest that the function of programming modules relies on synergistic interactions among transcription factors and thus cannot be extrapolated from the study of individual transcription factors in a different cellular context. In this study, we functionally characterize induced motor neurons that have been directly generated from ES cells via the forced expression of two different combinations of three transcription factors. Spinal motor neurons are induced via the expression of Ngn2, Isl1, and Lhx3 (iNIL), while cortical motor neurons are induced via the expression of Ngn2, Isl1, and Phox2a (iNIP). Here we profile the gene expression patterns of both types of induced motor neurons, directed differentiation motor neurons, and control cells. In all, 20 microarray experiments are provided in this submission, including 3 replicates of a control condition, 3 replicates of cells that have 24hrs induction of iNIL, 2 replicates of induced spinal motor neurons (induction of iNIL for 48hrs) that have been Hb9-GFP sorted, 3 replicates of induced spinal motor neurons exposed to retinoic acid that have been Hb9-GFP sorted, 3 replicates of motor neurons that have been differentiated in vitro using RA and Hh signalling, 3 replicates of induced cortical motor neurons (induction of iNIP for 48hrs), and 3 replicates of cells in which Isl1 in induced alone (induction of iI for 48hrs). For ChIP-Seq Samples: In this study, we functionally characterize induced motor neurons that have been directly generated from ES cells via the forced expression of two different combinations of three transcription factors. Spinal motor neurons are induced via the expression of Ngn2, Isl1, and Lhx3 (iNIL), while cortical motor neurons are induced via the expression of Ngn2, Isl1, and Phox2a (iNIP). The genome-wide binding of some of the programming factors is characterized here using ChIP-seq. We characterize the binding of Lhx3 and Isl1/2 in iNIL cells, Phox2a and Isl1/2 in iNIP cells, and Isl1/2 in cells in which Isl1 is induced alone (iI). There are 7 Illumina sequence datasets in this submission; one replicate for each of iLhx3-V5 and Isl1/2 in iNIL cells, two replicates for each of iPhox2a-V5 and Isl1/2 in iNIP cells, and one replicate for Isl1/2 in iI cells. An appropriate pseudo-IP control experiment is included.

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.

Dataset Information

CTCF functions as a chromatin insulator in the HoxA cluster during neurogenesis [RNA-Seq]

Publications

CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.

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