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. Author manuscript; available in PMC: 2013 Jun 3.
Published in final edited form as: Nature. 2011 Mar 20;472(7341):120–124. doi: 10.1038/nature09819

Figure 4. HOTTIP programs active chromatin via WDR5.

Figure 4

(A) Summary of RNA-protein interaction studies. Each of the indicated recombinant protein was purified and used to retrieve purified HOTTIP or control histone RNA in vitro. Only GST-WDR5 specifically retrieved HOTTIP. (B) HOTTIP binds directly and specifically to WDR5. Left: Purified GST and GST-WDR5 are visualized by SDS-PAGE and Comassie Blue staining. Right: Retrieved RNAs are quantified by qRT-PCR. (C) HOTTIP binds specifically to WDR5 in cells. IP of endogenous WDR5 protein from PC3 (prostate) and T24 (bladder) carcinoma cells specifically retrieved HOTTIP, but not control IPs with IgG or chromatin binder SIRT6. (D) WDR5 is required for 5′ HOXA gene expression, including HOTTIP. (E) HOTTIP recruitment potentiates transcription. Left: The BoxB tethering system. BoxB-RNA specifically binds λN fused to GAL4 DNA binding domain, recruiting the complex to a UAS-luciferase reporter gene. After transient transfection, IP of GAL4- λN specifically retrieves BoxB-HOTTIP. Right: Luciferase activity after co-transfection of the indicated constructs (mean ± s.d., n=4 triplicates, * indciates p<0.05 Student’s t-test comparing BoxB-LacZ vs. BoxB-HOTTIP). Sloped triangle indicates increasing input of plasmids encoding ncRNAs.