Fig. 4. De-repression of Hox genes in Rnf2^-/- MEFs and ES cells. (A) Conditional depletion of Rnf2 lead to de-repression of Hoxb8 in MEFs derived from the cranial part of Rnf2^fl/fl 9.5 dpc embryos. (Top) Infection of Cre-expressing adenovirus vector to MEFs derived from Rnf2^fl/fl embryos (fl/fl) depleted the Rnf2 gene products, whereas the wild-type (+/+) MEFs were unaffected. Lamin B was used as a control. (Bottom) The expression of Hoxb8 was induced by infection of Cre-expressing adenovirus vector in Rnf2^fl/fl MEFs (fl/fl), but not in the wild type (+/+). (B) Rnf2^-/- (-/-) ES cells were derived from Rnf2^fl/fl (fl/fl) ES cells by transient overexpression of Cre-recombinase. Rnf2 was re-expressed by transfecting Rnf2^-/- ES cells with a construct expressing Myc-tagged Rnf2 (tr). The expression of endogenous and transfected Rnf2 was examined by using anti-Rnf2 (left) and -Myc (middle) antibodies. CBB staining was used as a loading control (right). (C) The expression of Hox cluster genes in Rnf2^fl/fl (fl/fl), Rnf2^-/- (-/-) and Rnf2 transfected (tr) ES cells was compared by RT-PCR. The quantity of synthesized cDNA from respective cells was equalized by comparing the relative amounts of ß-actin transcripts. (D) The expression of Phc1 and Cbx2 gene products was reduced in Rnf2^-/- ES cells (-/-) in comparison with the wild type (fl/fl), whereas the expression of RYBP (another Rnf2-binding protein that is not found in hPRC-H complex or class 1 PcG proteins) was not altered. (E) Rnf2 association and H3-K27 trimethylation at Hox promoter regions were compared between Rnf2^fl/fl and Rnf2^-/- ES cells. For the `Input', genomic DNA extracted from the original whole cell lysate equivalent to the 1/40 volume of that used for the ChIP analysis was subjected to the PCR. Hprt was used as a negative control.