GSE122951
Ikaros is required for the controled activation of Notch target genes
Summary  Purpose: By means of high-throughput data analysis (high-throughput sequencing or next-generation sequencing -NGS-), we addressed the effect of the absence of Ikaros and the Notch pathway activation in mouse fetal liver erythroid cells. Specifically, the goals of this study are (i) to characterize the transcriptome of erythroid cells in the absence of Ikaros, when the Notch pathway is activated or not (by RNA-seq); (ii) to identify patterns in the modification of gene regulation according to the variable conditions; and (iii) to obtain information on mechanisms involved in the variation of gene regulation.    Methods: Erythroid cells obtained from Ikaros wild type (WT) or Ikaros knockout (Null) mouse fetal livers at the embryonic stage e14.5. These cells were co-cultured for 48 h on OP9 or OP9-DL1 cells.  mRNA profiles were generated by NGS, in biological triplicate, using Illumina cBot 2 System. The quality of the raw reads was assessed with FASTQC. Report show no pool imbalance. After examining the quality of the raw reads, no trimming was deemed necessary. The reads were aligned to the GRCm38/mm10 genome with TopHat. The raw alignment counts were calculated with htseq-count. DESeq2 calculates the differential expression of genes directly from the raw alignment counts calculated with htseq-count. The output from DESeq2 includes the raw counts normalized relative to the total number of reads. The log2 fold change is an estimate of the fold change between the conditions, based on the distribution of the reads. qRT-PCR validation was performed using specific primer sets in real-time PCR with SYBR Green.    Results. Based on expression profiles, all samples clustered correctly. The differential expression analysis was perform with all samples. For subsequent analysis, 2452 differentially expressed genes were identified in Ikaros WT vs Ikaros null cells cultured on OP9 cells, using a log2 = 1 and a p value <0.005. Additionally, 2847 differentially expressed genes were identified in Ikaros WT vs Ikaros Null cells cultured on OP9-DL1 cells, using a log2 = 1 and a p value <0.005. Among genes activated by Notch (cells cultured on OP9-DL1), 616 genes were repressed in the absence of Ikaros (Ikaros Null vs WT cells; log2= -0.8 and a p value <0.005) and 1558 genes were overexpressed in in the absence of Ikaros (Ikaros Null vs WT cells: log2= 0.8 and a p value <0.005), using adjusted p value <0.005. Modified expression or immunoprecipitation of several gene transcripts was confirmed with qRT-PCR.    Conclusions (Summary): The results obtained demonstrate that Ikaros can favour repression of Notch target genes but can also be required for proper activation on Notch targets upon Notch pathway activation. Overall design: Mouse e14.5 fetal liver erythroid cells mutated or not for Ikaros (Ikaros null allele) were cocultured on OP9 or OP9-DL1 cells for 48hours and used for RNA-sequencing. Biological triplicates were used.