Rapid and synchronous clearance of PcG histone modifications from Hox genes anticipates motor neuron differentiation
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Shaun Mahony
PROVIDER: E-GEOD-19450 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The expression of v5-tagged Hoxc9 is induced and ChIP-seq is used to profile genome-wide occupancy in differentiating motor neurons The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog signaling. Here, ChIP-seq is used to profile the genome-wide occupancy of Hoxc9 after five days of differentiation.
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Project description:Among its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating Retinoic Acid Receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites. We tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome RAR binds after initial differentiation. We have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of neurogenesis. Our data also suggests that enhancers active in pluripotent embryonic stem cells may be preselecting regions that will be activated by RAR during neuronal differentiation. The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid. Here, ChIP-seq is used to profile the genome-wide occupancy of RAR isofroms both immediately prior to and during exposure of the cells to retinoic acid. ChIP-seq is also used to profile the genomic occupancy of Pol2 with phosphorylated serine 5 (Pol2-S5P) and phosphorylated serine 2 (Pol2-S2P) after exposure to retinoic acid.
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Project description:Progenitor motor neurons can be generated with high-efficiency by differentiating ES cells in vitro in the presence of retinoic acid and hedgehog signalling. Here, we characterize the chromatin landscape associated with progenitor motor neurons (pMNs) in order to assess how histone modification domains shift during the differentiation process. In this study, we characterize the genomic occupancy of H3K27me3, H3K4me3, H3K79me2 and Pol2 using ChIP-seq in progenitor motor neurons that have been differentiated in vitro from ES cells. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included.
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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.
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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.
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