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. 2008 May 21;105(21):7472–7477. doi: 10.1073/pnas.0711896105

Fig. 5.

Fig. 5.

Ap2δ recruits Ash2l and Alr to the Hoxc8 locus. (A) Ap2δ, Ash2l, and Alr cooccupy specific regions of the Hoxc8 promoter located 27 bp (−27 bp) and 3 kb (−3.0 kb) upstream of the transcriptional start site. Chromatin fragments from Neuro2a cells expressing V5-Ap2δ were immunoprecipitated with anti-V5, -Ash2l, and -Alr antibodies. DNA association was analyzed by real-time PCR, using loci-specific primers. The heights of the bars represent means of fold increases for cells expressing V5-Ap2δ compared with wild-type control. The whiskers represent standard error of the mean. (B) (Top and Middle) Ap2δ down-regulation results in a significant decrease of Ash2l and Alr at the Hoxc8 promoter. Chromatin fragments from Neuro2a cells treated with either Tcfap2d-specific siRNA or scrambled control were immunoprecipitated with anti-Ash2l and -Alr antibodies. DNA association was determined by real-time PCR, and quantities are represented as ratios of signals relative to signals obtained from a nonspecific histone H3 control. The heights of the bars represent means of ratios for each condition performed in triplicate. The whiskers represent standard error of the mean. Statistical comparisons were made between conditions, using Student's t tests. *, P ≤ 0.05. (Bottom) Ap2δ down-regulation results in a significant decrease of H3K4me3 at the Hoxc8 promoter. Chromatin fragments from Neuro2a cells treated with either Tcfap2d-specific siRNA or scrambled control were immunoprecipitated with antibodies against H3K4me3. DNA analysis was performed in a similar manner as described for Top. (C) Alr and Ash2l colocalize at the −27/+161 region of the Hoxc8 promoter when Ap2δ is present. Chromatin fragments from NIH 3T3 cells, wild-type Neuro2a cells and Neuro2a cells expressing V5-Ap2δ were immunoprecipitated with anti-V5, -Alr, and -Ash2l antibodies. The immunoprecipitated DNA was analyzed by PCR, using locus-specific primers. NIH 3T3 cells, which do not express Ap2δ, were used as negative controls for the ChIP experiments. (D) Alr protein levels are unchanged when Ap2δ is down-regulated in Neuro2a cells. Total cell extracts were obtained from wild-type Neuro2a cells or Neuro2a cells treated with Tcfap2d-specific siRNA or scrambled control and analyzed by Western blot, using anti-Alr antibodies. (E) (Upper) Ap2δ and Ash2l regulate Hoxc8 expression in Neuro2a cells. Total RNA was extracted 72 h after transfection from Neuro2a cells treated with Tcfap2d- or Ash2l-specific siRNA or scrambled control. Gapdh and Hoxc8 transcript levels were quantified by real-time PCR. Normalized values were calculated as percentages of transcript levels detected in cells treated with the scrambled control. *, P < 0.001. (Lower) Knock down of either Ap2δ or Ash2l resulted in a significant decrease of their respective transcripts. Experiments were similar to those described in Upper.