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. 2010 Sep 20;190(6):1093–1106. doi: 10.1083/jcb.201002111

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© 2010 Bozec et al.

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Figure 5. — Oc and col1α2 are transcriptional target genes of Fra-2. (A) ChIP for Oc and Rankl promoter. Arrows indicate primers amplifying fragments. Chromatin of the indicated genotypes was immunoprecipitated with AP-1 antibodies. Quantification and endpoint qPCR-amplified fragments at 200 bp are shown. *, P < 0.01. (B) Coimmunoprecipitation for Fra-2 and ATF-4 in primary wt osteoblasts. IgG is used for loading control. Western blot analyses for 38 kD ATF-4 in Fosl2^+/+ (n = 2) and Fosl2^−/− (n = 3) long bones at P2. 42 kD actin is used as loading control. (C) H3K4me3 and H3K27me3 ChIP on Oc and Rankl promoter. Chromatin from osteoblasts of the indicated genotypes was immunoprecipitated with H3K4me3- and H3K27me3-specific antibodies. Bars represent qPCR quantification relative to input chromatin. (D) wt (pOG2-luc) or Ap-1 mutated (pOG2-MUT-luc) reporter assay for the Oc promoter fragment in the presence of ATF-4, Fra-2, ATF-4 + Fra-2, and ATF-4–Fra-2 expression vectors (n = 3). (E) ChIP for col1α2 promoter. Arrows indicate primers amplifying fragments at 200 bp. Chromatin of the indicated genotypes was immunoprecipitated with AP-1 antibodies. Endpoint qPCR-amplified fragments are shown. (F) H3K4me3 and H3K27me3 ChIP on col1α2 promoter. Chromatin from osteoblasts of the indicated genotypes was immunoprecipitated with H3K4me3- and H3K27me3-specific antibodies. Bars represent qPCR quantification relative to input chromatin. (G) pH5-luc, pH6-luc, and pH10-luc reporter assay for the col1α2 promoter fragment in the presence of Fra-2, c-Jun, JunB, c-Jun–Fra-2, and JunB–Fra-2 expression vectors (n = 3). Error bars represent SD. *, P < 0.01.