Figure 3.
Functional characterization of the IKZF1 susceptibility locus. (A) Epigenetic profile of the IKZF1 SNP rs58923657 susceptibility locus. Tracks show positions of rs58923657 and SNPs in LD (r2 ≥ 0.6); IKZF1; DNase I hypersensitive site clusters; GM12878 chromatin state (ChromHMM) corresponding to transcribed regions (green), candidate weak enhancers (yellow), or candidate strong enhancers (orange); and ChIP-seq data for H3K4Me1, H3K4Me3, H3K27ac and transcription factors from ENCODE.36 Data are displayed in the UCSC genome browser (http://genome.ucsc.edu/)46 and the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/).47 (B) Chromatin spatial organization of chromosome 7 from 50.0-50.6 MB. Tracks show a Hi-C heatmap of chromatin contact frequencies in GM12878; chromatin contact domain determined by the Arrowhead algorithm38; CD19+ B-cell superenhancers37; canonical transcripts; CTCF ChIP-seq data in GM12878 from ENCODE; and CTCF ChIA-PET mapping data in GM12878.39 Black rectangle indicates the rs58923657 locus. (C) Risk and nonrisk allele-specific enhancers encompassing rs58923657 were cloned into the pGL3-promoter vector (Promega) and transfected into 20 LCLs (10 DS and 10 non-DS). Enhancer reporter assay demonstrates relative luciferase activity measured 24 hours later for the nonrisk and risk alleles relative to the empty vector. Bars show the mean ± SEM from transfections performed in triplicate in the 20 LCLs. (D) EMSAs were performed using nuclear extracts from 6 patient-derived LCLs (3 DS and 3 non-DS) reacted with double-stranded DNA probes encompassing the indicated IKZF1 SNPs. Bars show mean ratios ± SEM of risk to nonrisk allele protein binding. (E) IKZF1 knockdown results in increased cellular proliferation with a greater effect in DS LCLs. Serial cell counts demonstrated significantly greater cellular proliferation for 4 DS LCLs expressing IKZF1-shRNA compared with NT-shRNA (P = .026) and 4 non-DS LCLs expressing IKZF1-shRNA compared with NT-shRNA (P = .017), and the effect of IKZF1-shRNA was significantly greater in the DS LCLs than in the non-DS LCLs (P = .047). Data shown are means of experiments performed in triplicate. P values in panels C-E were determined by a Student 2-tailed t test. *P < .05; **P < .001; ***P < .0001.

Functional characterization of the IKZF1 susceptibility locus. (A) Epigenetic profile of the IKZF1 SNP rs58923657 susceptibility locus. Tracks show positions of rs58923657 and SNPs in LD (r2 ≥ 0.6); IKZF1; DNase I hypersensitive site clusters; GM12878 chromatin state (ChromHMM) corresponding to transcribed regions (green), candidate weak enhancers (yellow), or candidate strong enhancers (orange); and ChIP-seq data for H3K4Me1, H3K4Me3, H3K27ac and transcription factors from ENCODE.36  Data are displayed in the UCSC genome browser (http://genome.ucsc.edu/)46  and the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/).47  (B) Chromatin spatial organization of chromosome 7 from 50.0-50.6 MB. Tracks show a Hi-C heatmap of chromatin contact frequencies in GM12878; chromatin contact domain determined by the Arrowhead algorithm38 ; CD19+ B-cell superenhancers37 ; canonical transcripts; CTCF ChIP-seq data in GM12878 from ENCODE; and CTCF ChIA-PET mapping data in GM12878.39  Black rectangle indicates the rs58923657 locus. (C) Risk and nonrisk allele-specific enhancers encompassing rs58923657 were cloned into the pGL3-promoter vector (Promega) and transfected into 20 LCLs (10 DS and 10 non-DS). Enhancer reporter assay demonstrates relative luciferase activity measured 24 hours later for the nonrisk and risk alleles relative to the empty vector. Bars show the mean ± SEM from transfections performed in triplicate in the 20 LCLs. (D) EMSAs were performed using nuclear extracts from 6 patient-derived LCLs (3 DS and 3 non-DS) reacted with double-stranded DNA probes encompassing the indicated IKZF1 SNPs. Bars show mean ratios ± SEM of risk to nonrisk allele protein binding. (E) IKZF1 knockdown results in increased cellular proliferation with a greater effect in DS LCLs. Serial cell counts demonstrated significantly greater cellular proliferation for 4 DS LCLs expressing IKZF1-shRNA compared with NT-shRNA (P = .026) and 4 non-DS LCLs expressing IKZF1-shRNA compared with NT-shRNA (P = .017), and the effect of IKZF1-shRNA was significantly greater in the DS LCLs than in the non-DS LCLs (P = .047). Data shown are means of experiments performed in triplicate. P values in panels C-E were determined by a Student 2-tailed t test. *P < .05; **P < .001; ***P < .0001.

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