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. 2013 Mar 1;32(7):982–995. doi: 10.1038/emboj.2013.37

Figure 4.

Figure 4

HLX1 binds to the INK4a promoter. (A) HLX1 regulates INK4a mRNA levels. IMR-90 cells were infected with pRS-shHLX1.2 or a control pRS vector and the levels of INK4a mRNA were evaluated by qRT–PCR. (B) Luciferase reporter assay showing that HLX1 expression prevents the activation of the INK4a promoter by ETS2. (C) Cartoon depicting the INK4/ARF locus and the primer sets used for ChIP. Primer sequences are in Supplementary Table S3. (D) HLX1 binds in the proximity to the INK4a promoter, as assessed by ChIP from IMR-90 cells infected with pBABE-HLX1. Chromatin from IMR-90 cells at passages 24–25 was used for these experiments. Binding of HLX1 to primer sets 5 and 6 was significant. (E) Cartoon depicting the INK4a/ARF locus showing the Homeobox binding domains (blue), the regions amplified by the ChIP primer sets 5 and 6, and the probes used for the DNA pulldown experiments. (F, G) DNA pulldown assays showing HLX1 binding to probes corresponding to the ∼−850 (F) and∼−550 (G) Homeobox binding domain regions. Sequences of oligos used for the probes are included in Supplementary Table S4. wt, wild type; m, mutant. **P<0.01.