Figure 5: Functional exploration of rs1247117 in B-ALL cells.

(A) IGV genome browser image in Nalm6 cells showing the genomic context, chromatin accessibility, and EIF3A promoter connectivity using promoter CHiC of the top functional regulatory variant, rs1247117, with the highest allele-specific MPRA activity. Genomic binding profiles are also shown for RNA polymerase II (RNA Pol2), histone H3 lysine 27 acetylation (H3K27Ac) and PU.1. (B) rs1247117 lies in a PU.1 binding motif. The human genome reference sequence, Nalm6 genome sequence, location of rs1247117 and PU.1 position weight matrix is shown. (C) Design of biotinylated DNA-probes for in vitro rs1247117 pulldown. (D) Biotinylated DNA pulldown shows rs1247117 allele-dependent enrichment of PU.1 binding. Blot shown is representative of two independent experiments. P-value from densitometric quantification of two blots is shown. (E) Diagram on the left showing the genomic context of the rs1247117 CRE deletion in Nalm6 cells in relation to chromatin accessibility, PU.1 binding and rs1247117. Black bar represents ATAC-seq peak, green par represents PU.1 peak, and red bar represents region deleted using CRISPR/Cas9 genome editing. Gel shows validation of deletion using primers flanking deleted region. Arrow points to PCR fragment with deletion in heterogeneous Nalm6 cell pools harboring deletion compared to wild-type parental Nalm6 cells. (F-G) CACUL1 (F) and EIF3A (G) expression is upregulated upon deletion of the CRE containing rs1247117. RT-qPCR data show the mean +/− SEM of three independent experiments. (H) Drug sensitivity data comparing survival of wild-type parental Nalm6 cells and Nalm6 cells with rs1247117 CRE deletion after vincristine (VCR) treatment for 24 (n=3), 48 (n=3) and 72 (n=3) hours. Vincristine concentration is provided below. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data show the mean +/− SEM relative to untreated cells.