Unknown,Transcriptomics,Genomics,Proteomics

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Msx1 ChIP-Seq from C2C12 murine myoblast cells expressing Flag-Msx1 protein

ABSTRACT: The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. We used genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1 in C2C12 murine myoblast cells. C2C12 myoblast cells were seeded at a density of 2.5 x 10^5 cells / 10-cm plate one day prior to infection, and infected with a retrovirus containing a Flag-Msx1 construct for two consecutive days. The resulting cells were then crosslinked with formaldehyde, and DNA was enriched by chromatin immunoprecipitation (ChIP) with an anti-Flag antibody and analyzed by Solexa sequencing. Enriched regions were identified using a Poissonian background model, and were further compared to an additional background of sequences from a sample of Flag-immunoprecipitated DNA from C2C12 cells infected with an empty vector to determine enrichment. ChIP was performed using an antibody against the Flag epitope (Sigma M2, F3165).

ORGANISM(S): Mus musculus

SUBMITTER: Richard Young

PROVIDER: E-GEOD-26711 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. Using genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1, we find that repressed target genes are enriched at the nuclear periphery in myoblast cells. We further show that the interaction of Msx1 with the Polycomb repressive complex PRC2 is required for transcriptional repression and regulation of myoblast differentiation, and promotes increased tri-methylation of lysine 27 on histone H3 (H3K27me3) at Msx1 target genes. Furthermore, Msx1 genomic binding promotes the dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in developing embryos in vivo. Thus, our findings suggest a hitherto unappreciated spatial coordination of transcription factor binding, Polycomb recruitment, and subnuclear localization in regulation of developmentgene expression programs. In order to identify genes regulated by Msx1, we infected C2C12 myoblast cells with a retrovirus expressing a tamoxifen-regulated Msx1 protein, Msx1-ER (or with empty vector as a control), followed by induction with 0.2 nM of tamoxifen or vehicle (DMSO) for 6 hours. Regulated genes were identified as those that changed in expression upon tamoxifen induction of the Msx1-ER protein but did not change in the empty vector control. Experiments were performed in triplicate for each of the four experimental conditions (Msx1-ER + tamoxifen, Msx1-ER - tamoxifen, empty vector control + tamoxifen, empty vector control - tamoxifen), for a total of 12 independent array samples. This submission represents the transcriptome component of the study.

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Msx1 ChIP-Seq from C2C12 murine myoblast cells expressing Flag-Msx1 protein

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