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. 2016 Apr 20;15(11):1479–1493. doi: 10.1080/15384101.2016.1175797

Figure 5.

Figure 5.

The Ring1b-Med12 target gene Hoxd11 is regulated by the ncRNA Dlx1as. (A) Med12 and Cbx7-PRC1 but not Cdk8, localize to Hoxd11 in mESCs. IGV Screenshot from RNAseq (mESCs) and ChIP-seq peaks illustrating the binding of Med12, Med1, Cdk8, Ring1b, Cbx7, RYBP and Suz12 at the Hoxd11 locus. (B) Illustration of the 2 Mb locus spanning the Dlx1as gene (green) and the HoxD cluster (red). The direction of transcription is indicated by the arrowheads. (C) The ncRNA Dlx1as regulates the expression of genes of the HoxD cluster in cis. mESCs were transfected with control siRNA or siRNA directed against Dlx1as and treated with RA. Twenty-four hours after RA administration the relative expression of genes in the 2 Mb locus were analyzed by qRT-PCR. Data are represented as a mean +/− SEM (n = 3). *** P-value<0.0001, ** P-value <0.01, * P-value < 0.02 as calculated by 2-tailed t test. (D) Med12 associates with Dlx1as in pluripotency and early differentiation. RIP experiments with Med12 antibodies and control IgGs from extracts of mESCs and differentiating cells (24h RA). The association of Dlx1as was analyzed in qRT-PCR experiments. Data are represented as a mean +/− SEM (n = 3). (E) Med12 associates with Dlx1as as a function of Ring1b. RIP experiments with Med12 antibodies and control IgGs using extracts from control and Ring1b knockdown mESCs. The association of Dlx1as, Hotairm (linc1548) and Rian was analyzed in qRT-PCR experiments. Data are represented as a mean +/− SEM (n = 3). The indicated P-value was calculated by F test to compare the differences. (F) Ring1b dissociates from Dlx1as upon differentiation. RIP experiments with Ring1b antibodies and control IgGs from extracts of mESCs and differentiating cells (24h RA). The association of the indicated ncRNAs Dlx1as, Hotairm and Rian was analyzed in qRT-PCR experiments. The figure represents an average of at least 3 independent experiments +/− SEM. The indicated P-value was calculated by F test to compare the differences. (G) The association of Ring1b and Med12 is RNA-mediated. Endogenous Ring1b IPs with nuclear extracts from wt mESCs. Precipitated material was subjected to RNaseA treatment prior to Western blotting and incubated with the indicated antibodies. Inputs represent 5% of the material used for the IPs. The relative intensity of Med12 Co-IP levels was calculated using the Ring1b levels as reference.