genome-wide-snapshot-chromatin-regulators-states-xenopus-embryos-chip

Genome-wide Snapshot of Chromatin Regulators and States in <em>Xenopus</em> Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

MRC National Institute for Medical Research

The design of effective cell replacement therapies requires detailed knowledge of how embryonic stem cells form primary tissues, such as mesoderm or neurectoderm that later become skeletal muscle or nervous system. Members of the T-box transcription factor family are key in the formation of these primary tissues, but their underlying molecular activities are poorly understood. Here, we define in vivo genome-wide regulatory inputs of the T-box proteins Brachyury, Eomesodermin, and VegT, which together maintain neuromesodermal stem cells and determine their bipotential fates in frog embryos. These T-box proteins are all recruited to the same genomic recognition sites, from where they activate genes involved in stem cell maintenance and mesoderm formation while repressing neurogenic genes. Consequently, their loss causes embryos to form an oversized neural tube with no mesodermal derivatives. This collaboration between T-box family members thus ensures the continuous formation of correctly proportioned neural and mesodermal tissues in vertebrate embryos during axial elongation.

In vivo T-box transcription factor profiling reveals joint regulation of embryonic neuromesodermal bipotency.

Chromatin immunoprecipitation (ChIP) combined with genomic analysis techniques provide a global snapshot of protein-DNA interactions in the context of chromatin, yielding insights into which genomic loci might be regulated by the DNA-associated protein under investigation. This protocol describes how to perform ChIP on intact or dissected Xenopus embryos. The ChIP-isolated DNA fragments are suitable for high-throughput sequencing (ChIP-Seq) or for quantitative PCR (ChIP-qPCR). In this protocol, embryonic tissue is harvested from Xenopus tropicalis or Xenopus laevis at the developmental stage of interest, and DNA-associated proteins are immobilized to their endogenous genomic binding sites with formaldehyde. Nuclei are extracted from embryos and subjected to sonication so as to shear the chromatin to a size that allows sufficient positional resolution of protein binding to genomic DNA. Chromatin fragments bound by the protein of interest are immunoprecipitated using antibody-coupled beads, washed under high-stringency conditions, and stripped from the beads with anionic detergents. The chemical cross-links are reversed, and the coimmunoprecipitated DNA is purified. The resulting DNA fragments can be analyzed by qPCR or used to create a ChIP-Seq library. General advice for qPCR and for making ChIP-Seq libraries is offered, and approaches for analyzing ChIP-Seq data are outlined.

George E. Gentsch

Division of Systems Biology

MRC National Institute for Medical Research

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

George E. Gentsch

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MRC National Institute for Medical Research

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Division of Systems Biology