Fig. 6

Transcriptional regulation of ARL4D by subtype-specific SE and SE-associated TF in G3-MB. a Gene tracks of H3K27Ac ChIP-seq signal across G3-MB cells and tissues at proximal SE regions of ARL4D. SE region is depicted in colored line over the gene tracks. The gene track images were obtained from https://viz.stjude.cloud/st-jude-childrens-research-hospital/visualization/chip-seq-landscape-of-primary-medulloblastomas~23. b Gene tracks of H3K27Ac ChIP-seq signal across five G3-MB lines and non-G3-MB line UW228 at proximal SE regions of ARL4D. SE regions are depicted in colored lines over the gene tracks. The positions of predicated binding motifs of OTX2, ChIP-qPCR amplicons, pooled-CRISPRi sgRNA targeting regions and 3C-PCR primers are shown below the tracks. c ChIP-qPCR analysis of H3K27Ac (top) and OTX2 (bottom) enrichment near ARL4D in MB002, D425 and UW228. Tested positions of amplicons are shown in (a). d 3C-PCR analysis of chromatin interaction between the H3K27Ac peak regions within SE and promoter of ARL4D in MB002 and UW228. The workflow of 3C-PCR was shown above the agarose gel electrophoresis of 3C-PCR products. Positions of 3C-PCR primers are shown in a. e Pooled-CRISPRi sgRNAs are designed to target the SE regions of ARL4D in stable dCas9-KRAB expressing MB002, the ARL4D expression level (left) is measured by RT-qPCR, and cell viabilities (right) are measured at Day 6. The pooled-CRISPRi sgRNA targeting regions are shown in (a). f ChIP-qPCR analysis of H3K27Ac enrichment near ARL4D in MB002 treated with 2 μM JQ1 or 0.05 μM THZ1 for 24 h, respectively. All cell viability, ChIP-qPCR and RT-qPCR assays were performed in triplicate and the data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (c and f) and two-tailed t test (e)