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. Author manuscript; available in PMC: 2018 Jul 10.
Published in final edited form as: Dev Cell. 2017 Jul 10;42(1):52–67.e4. doi: 10.1016/j.devcel.2017.06.009

Figure 6. THAP1 Colocalizes With and Affects YY1 Occupancy.

Figure 6

(A) Schematic of the strategy used to identify putative co-factors for THAP1. The genomic regions corresponding to TSS (±1kb) of the 44 THAP1-CNS genes (genes bound and regulated by THAP1) were subjected to independent analyses to identify TF with significantly enriched transcription factor binding (ENCODE CHIP-Seq Significance Tool) and significantly enriched DNA binding motifs (Distant Regulatory Elements (“DiRE”). Eight genes were identified in common in these analyses, which we define as putative THAP1-cofactors.

(B, D–G) THAP1 regulates YY1 occupancy in OL. (B) Genome Browser track (IGV) showing CHIP-Seq signals for THAP1 and YY1 for 5 candidate genes (Ech1, Cuedc2,Prepl, Dars2, Tmem180) and 2 control genes (Gapdh and Hprt). (D–E) Quantitative CHIP (qCHIP) demonstrating specificity for antibodies (Goat anti-THAP1 and Rabit anti-YY1) used in CHIP studies. Binding, represented as % input (Y-axis) demonstrated for (D) THAP1 (E) YY1 and their respective isotype control IgG (Goat IgG or Rabbit IgG) for candidate genes (Ech1, Cuedc2, Gapdh and Hprt) tested (x-axis). The y-axis (% input) represents the final amount of immunoprecipitated chromatin/gene assessed as the percentage of total input chromatin from corresponding control (Thap1+/+) and Thap1 null ES cells (Thap1−/−) (Experimental Procedures). (F–G) qCHIP demonstrating binding represented as % input (y-axis) for THAP1 (E) and YY1 (F) in control Thap1 null OL cells at the 22 gene loci tasted (x-axis). Significance value was determined using T-test (* = p < 0.05, ** = P < 0.01 and *** = p<0.001).

(C) Coimmunoprecipitation demonstrating THAP1 interactions with YY1. HEK-293T cells transfected with YY1 and THAP1-FLAG1 was immunoprecipitated with goat anti-THAP1, rabbit anti-YY1 or normalized rabbit IgG and immunobloted with anti-FLAG, YY1 or rabbit anti-THAP1.

(H) Id genes are selectively upregulated in Thap1 null OL. qRT-PCR is depicted for multiple days (x-axis) following PDGF withdrawal for candidate genes regulated by BMP pathway. Gene expression values are normalized to Olig2 expression and represented as fold change (y-axis) compared to Day 0 OPC values. 2-way ANOVA demonstrates a main effect of genotype (P<0.01) for all genes shown in panel G. (*) represents time points where significant differences exist using post hoc Sidak’s multiple comparison test. Note that the expression of Thap1 in (H) is reproduced (5I) for clarity of comparisons in differentiation gene expression.