Figure 6
The XLOC_000895/Robnr locus regulates Bcl2 expression. (A) Genome browser tracks showing the RNA-seq and ChIP-seq data on the XLOC_000895/Robnr locus. The CRISPR/Cas9 targeted region is highlighted in orange. The location of guide RNAs (gRNAs) are denoted by vertical blue lines and the size of the targeted region is indicated. The primers used for RT-qPCR analysis are denoted by vertical red lines. (B) Relative expression levels of Bcl2 and Robnr at different time points of the PMA/ionomycin treatment in wild-type (wt) and Robnr-mutated clones, ΔRobnr-cl1 and ΔRobnr-cl2. Rpl32 was used for normalization. The statistical significance was assessed at 4 h of stimulation from 3 biological replicates by a Student’s t-test (unpaired, one-tailed; ***P < 0.001, **P < 0.01, *P < 0.1). Data are represented with standard deviation. Details about the gRNA sequences, the PCR primers and the expected amplicons are provided in Supplementary Table 1. (C) Genome browser tracks showing the RNA-seq and ChIP-seq data surrounding the Bcl2 promoter. Three genomic regions displaying a higher H3K4me3 enrichment in PMA/ionomycin stimulated cells are highlighted. The primers used for ChIP validation are denoted by vertical blue lines. (D) ChIP-qPCR analysis of the H3K4me3 enrichment on the Bcl2 promoter in wild-type (wt) and the two Robnr-mutated clones, after an exposure to DMSO or PMA/ionomycin (PMA/Iono) for 4 h. The graph shows the results of 3 replicates. The promoter of Actb was used for normalization.
