Study of developmentally regulated transcription factors by chromatin immunoprecipitation and sequencing (ChIP-seq) faces two major obstacles: availability of ChIP grade antibodies and access to sufficient number of cells. that govern cell fate PETCM specification and embryonic development. While numerous transcription factors controlling cell differentiation have been functionally characterized their cell type specific patterns of DNA binding remain largely unknown. The method of choice for genome-wide mapping of transcription factor binding sites is chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) 1. Although powerful current ChIP-seq technology is limited by two critical factors when applied to developmental studies. First ChIP-seq profiling demands a large number of cells (20-50 million) separated from PETCM PETCM other cell types expressing the transcription factor of interest and second it requires antibodies with high affinity and specificity that recognize transcription factors in their native form bound to DNA. To overcome these two hurdles we combined a versatile system for generating mouse embryonic stem cell (ESC) lines harboring inducible and epitope-tagged transcription factors with directed differentiation of ESCs along defined cellular lineages. This system presents several advantages: 1) the use of tagged transcription factors or DNA binding proteins obviates the need for validated factor-specific antibodies; 2) the use of pluripotent cells allows analysis of any developmental cell lineage; and 3) the inducible expression makes it possible to examine binding of developmentally regulated transcription factors in their correct developmental context as well as to study tagged transcription factors by gain-of-function analysis. To overcome the inconsistency and inefficiency of classical transgenic ESC line production we relied on a recently developed inducible cassette exchange (ICE) system 2. The resulting transgenic lines harbor a single copy of the transgene recombined into a defined expression-competent locus. To further streamline the generation of inducible cell lines we introduced Gateway (Invitrogen) landing sites into the shuttle vector and a short epitope tag either at the amino- (Flag-Bio) or carboxy-terminus (His-V5) of the protein (Fig. 1a). Because of the high efficiency of all actions parallel production of multiple inducible tagged lines can be accomplished in as little as three weeks. Physique 1 Generation of inducible cell lines Differentiation of mouse ESCs to spinal motor neurons yields scalable and largely homogeneous populations of cells mirroring developmentally relevant motor neuron differentiation says in mouse 3. To test our approach we first investigated genome-wide binding of the bHLH transcription factor Olig2 in motor neuron progenitors (pMNs) 4 a rare PETCM population of cells (<1% of spinal cells on e9.5) found in the embryonic ventral spinal cord 5. We generated an inducible Olig2 ESC range where Olig2 proteins is certainly carboxy-terminal tagged using the V5 epitope (iOlig2-V5). To imitate the standard Olig2 design of appearance doxycycline (Dox) was implemented late on Time 3 as well as the expression from the transgene was examined on Time 4 (Fig. 1b) when cells reach pMN stage. PETCM The transgenic Olig2-V5 proteins was portrayed uniformly in pMNs exhibited appropriate nuclear localization and it is expressed at amounts ~4 fold greater than indigenous Olig2 (Suppl. Fig. 1a-b). The V5 series didn't perturb the function from the tagged Olig2-V5 proteins. Needlessly to say ectopic appearance of Olig2-V5 led to the repression of Nkx2.2 in ventral interneuron progenitors (Fig. 1c) 4 and in the repression of Pax6 and Irx3 in dorsal interneuron progenitors (Fig. 1d) 6. As a result a tagged edition of Olig2 recapitulates in differentiating Rabbit polyclonal to IFIT2. ESCs the standard function of indigenous Olig2 during spinal-cord advancement 7. To account Olig2 binding we induced Olig2-V5 in pMNs and performed a ChIP-seq test out an anti-V5 antibody. We noticed that Olig2-V5 binds in the closeness from the downregulated genes Irx3 Nkx2.2 and Pax6 (Fig 2a and Suppl. Fig. 1c) indicating that Olig2 specifies pMN identification by immediate repression of interneuron transcriptional applications. Figure 2 Local and tagged ChIP evaluations The overexpression from the Olig2.