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The interplay of Ascl1 and Zfp238 and other targets is yet another aspect of the hierarchical organization of iN cell reprogramming. First, based on binding and transcriptional data Ascl1 is the direct upstream activator of Zfp238. Second, Zfp238 executes an important but only a part of the lineage-specification program of Ascl1, as we found three additional VX-770 manufacturer transcription factors that cooperate with Zfp238 to generate iN cells. Hence, out of the thousands of target genes, only a handful appears to be critical for neuronal reprogramming. This conclusion may be helpful to enable reprogramming of thus far resistant cell types, such as keratinocytes. Collectively, our results suggest that iN cell reprogramming is a powerful tool to interrogate transcription factor function and to uncover transcriptional networks in neuronal lineage specification. TauEGFP MEFs were isolated, and lentivirus was produced as previously described (Vierbuchen et?al., 2010). ES cell-derived NPCs (line NS5) were obtained from A. Smith, Cambridge, UK, and cultured as previously reported (Conti et?al., 2005). To establish Ascl1-inducible www.selleckchem.com/products/ch5424802.html MEFs, we first generated a Rosa26 CAGGS-M2rtTA-T2A-puro ES cell line by homologous recombination and infected this with a TetO-flagAscl1 lentivirus. A subclone showing robust induction was then injected into blastocysts to generate chimeric embryos for MEF isolation. More detailed information can be found in the Extended Experimental Procedures. Total RNA was isolated and Poly-A selected. Libraries were prepared, and sequencing reads (100?bp) were generated on Hi-Seq 2000 Illumina platforms. Paired-end reads were aligned to the mouse reference sequence NCBI Build 37/mm9. Expression levels of RefSeq-annotated genes were calculated in unit of fragments per kilobase of exon model per million mapped fragments (FPKM). Differential expression analysis was performed using Student��s t test function ��t.test�� in R, and genes with a p value?Ribonucleotide reductase as significant. Gene ontology analysis was performed using DAVID (david.abcc.ncifcrf.gov). ChIP-qPCR and ChIP-seq were carried out in Ascl1- and BAM-infected MEFs 48?hr after dox, and in uninfected or Ascl1-infected NPCs 18?hr after induction. Cells were infected with a pool of Ascl1, V5- or FLAG-tagged Brn2, and V5- or FLAG-tagged Myt1l, and the transgenes were induced with dox the day after. A list of antibodies used and detailed experimental procedures, including ChIP co-IP and primary computational analysis, can be found in the Extended Experimental Procedures. Formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) followed the previously described method (Giresi et?al., 2007). To identify chromatin states, we used the ChromHMM software (v. 1.