Fig 2: TAF9B binds promoter and distal regions of neuronal genes.(A) TAF9B, OLIG2, HOXC9 and RNA POL2 ChIP-seq peaks examples are given for Tubb3, Mnx1, Ngn2 and Hes6 genes. (B) Total numbers of peaks obtained by MACS in each ChIP-seq category. (C) ChIP-seq analysis of TAF9B and RNA POL2 binding sites in EB-MN samples relative to their distance to annotated transcription start sites (TSS) performed using GREAT. (D) Percentage of genes affected more than twofold in the absence of TAF9B as determined by RNA-seq analysis in Taf9b KO EB-MN differentiated for 6 days. Genes are grouped in different categories depending on the distance of the TAF9B peaks from their TSS. Genes associated to TAF9B-only peaks were used for the analysis. A gene list randomly generated was used as control. (E) Representative ChIP-qPCR analysis of TAF9B and RNA POL2 of selected loci identified by ChIP-seq. D = distal region, P = proximal promoter, NC = negative control. Bars represent mean ± SD of three replicates.DOI: http://dx.doi.org/10.7554/eLife.02559.010

Fig 3: TAF9B is required for efficient differentiation of murine ES cells into motor neurons in vitro.(A) qRT-PCR analysis for the MN markers (Mnx1, Lhx3, Isl1) and ES cell markers (Pou5f1 and Nanog) in WT and Taf9b KO cells differentiated for 6 days. Values are relative to undifferentiated ES cells. Bars show mean ± SEM of three biological replicates. (B) WT and Taf9b KO EB-MN, differentiated for 6 days, were immunostained using antibodies against MN marker ISL1/2, ES cell markers OCT4 and NANOG, and cellular proliferation marker Ki67. (C) Western blot analysis of WT and Taf9b KO EB-MN samples differentiated for 6 and 8 days detecting neuronal marker TUBB3 and ACTB. (D) Apoptosis levels were determined using an annexin V based assay in WT and Taf9b KO cells at different time points during MN differentiation. Graph shows mean ± SEM for the ratio of Taf9b KO to WT cells in three biological replicates.DOI: http://dx.doi.org/10.7554/eLife.02559.006

Fig 4: TAF9B controls neuronal gene expression in vivo.(A) Taf9b KO ES cells were used to generate a Taf9b chimeric mouse which was subsequently mated with WT to test for germline transmission and obtain Taf9b heterozygous mice. (B) Representative PCR-based DNA genotyping of Taf9b KO mice. Primers are described in Supplementary file 3. (C) Number of pups and weight of newborn mice from WT × WT and KO × KO matings. (D) RNA-seq data of spinal cord sample for Taf9b locus in WT and Taf9b KO animals. (E) Gene expression analysis by qRT-PCR of lumbar spinal columns tissues of newborn mice. Graphs represent mean ± SEM of littermate comparisons (WT n = 6, KO n = 8).DOI: http://dx.doi.org/10.7554/eLife.02559.013

Fig 5: TAF9B binds promoter and distal regions of neuronal genes.(A) ChIP-seq analysis of TAF9B and RNA POL2 binding sites in EB-MN samples differentiated for 8 days. TAF9B and RNA POL2 ChIP-seq peaks examples are given for Tubb3 and Hes6 genes. (B) List of top GO terms Biological Process of genes associated to TAF9B distal peaks and TAF9B peaks at annotated transcription start sites (TSS) ranked by p-value. Association of ChIP-seq peak to annotated genes and GO analysis was performed using GREAT. (C) Co-localization of TAF9B distal peaks and TAF9B peaks at TSS with OLIG2 ChIP-seq peaks in EB-MN samples.DOI: http://dx.doi.org/10.7554/eLife.02559.009