Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Induced Cdx2 binding in progenitor motor neurons and its effect on H3K27me3 chromatin domains [Affymetrix]

ABSTRACT: We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. Expression studies: Affymetrix arrays are used to profile gene expression in ES cells, RA/Hh-derived Day 5 motor neurons, and RA/Hh-derived motor neurons that have also been exposed to Dox (to activate iCdx2) and FGF.

ORGANISM(S): Mus musculus

SUBMITTER: Shaun Mahony

PROVIDER: E-GEOD-39422 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

Project description:We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. ChIP-seq studies: We characterize the binding of Cdx2 in progenitor motor neurons using a V5 tagged doxycycline inducible Cdx2 ESC line (iCdx2). Progenitor motor neurons were generated after 4 days of in vitro differentiation of mouse embryonic stem cells using retinoic acid (RA) and hedgehog (Hh) signaling exposure at day 2. On day 3, the cells are exposed to Dox with and without accompanying FGF signaling. The genome-wide binding of the induced Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 antibody. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included. We also examine the effect of induced Cdx2 expression on polycomb-associated chromatin structure in the resulting cellular populations by profiling the H3K27me3 chromatin mark using ChIP-seq. H3K27me3 experiments were performed after 5 days of in vitro differentiation using cells exposed to either: 1) RA & Hh to derive progenitor motor neurons, followed by Dox & FGF; 2) Dox & FGF alone; or 3) RA and Hh alone. There are 6 Illumina sequencing datasets included in this submission: two biological replicates of iCdx2 ChIP-seq in the presence of FGF; one sample of iCdx2 ChIP-seq in the absence of FGF; one H3K27me3 ChIP-seq in the presence of RA, Hh, Dox, and FGF; one H3K27me3 ChIP-seq in the presence of Dox and FGF; and one H3K27me3 ChIP-seq in the presence of RA and Hh.

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:Regulatory proteins can bind to different sets of genomic targets in various cell types or conditions. To reliably characterize such condition-specific regulatory binding we introduce MultiGPS, an integrated machine learning approach for the analysis of multiple related ChIP-seq experiments. MultiGPS is based on a generalized Expectation Maximization framework that shares information across multiple experiments for binding event discovery. We demonstrate that our framework enables the simultaneous modeling of sparse condition-specific binding changes, sequence dependence, and replicate-specific noise sources. MultiGPS encourages consistency in reported binding event locations across multiple-condition ChIP-seq datasets and provides accurate estimation of ChIP enrichment levels at each event. MultiGPSM-bM-^@M-^Ys multi-experiment modeling approach thus provides a reliable platform for detecting differential binding enrichment across experimental conditions. We demonstrate the advantages of MultiGPS with an analysis of Cdx2 binding in three distinct developmental contexts. By accurately characterizing condition-specific Cdx2 binding, MultiGPS enables novel insight into the mechanistic basis of Cdx2 site selectivity. Specifically, the condition-specific Cdx2 sites characterized by MultiGPS are highly associated with pre-existing genomic context, suggesting that such sites are pre-determined by cell-specific regulatory architecture. However, MultiGPS-defined condition-independent sites are not predicted by pre-existing regulatory signals, suggesting that Cdx2 can bind to a subset of locations regardless of genomic environment. In this study, we characterize the binding of Cdx2 in embryonic stem cells, endodermal cells, and progenitor motor neurons using V5- or FLAG-tagged doxycycline inducible Cdx2 ESC lines (iCdx2). Endoderm and progenitor motor neurons are generated from the ES cells using directed differentiation approaches. The cells are then exposed to Dox to express the tagged Cdx2 construct. The genome-wide binding of the induced full-length Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 or anti-FLAG antibody. We also examine the binding behavior of a truncated version of the Cdx2 protein, where a protein interaction domain contained in the first 59 amino acids has been deleted. An appropriate pseudo-IP control experiment for these ChIP-seq experiments has been previously submitted under accession number GSM766062.

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.