Figure S5.

A Model Cell System to Inducibly Disrupt Targeting of the PCGF1/PRC1 Complex, Related to Figure 5

(A) A schematic of the Kdm2b gene showing the long form (Kdm2b-LF) and short form (Kdm2b-SF) transcription start sites. The positions of LoxP sites are highlighted flanking the exon which encodes the ZF-CxxC domain.

(B) PCR with primers spanning the floxed exon was performed on genomic DNA from the Kdm2b^fl/fl cells (UNT), the Kdm2b^fl/fl cells treated for 72 hr with tamoxifen (72 hr), and wild-type cells (WT). 72hrs of tamoxifen treatment leads to a clear deletion of the floxed exon.

(C) Western blot analysis indicates that loss of the KDM2B ZF-CxxC domain does not lead to destabilization of the PCGF1 and RING1B components of the KDM2B variant PRC1 complex or upregulation of its paralogue KDM2A. Furthermore, PRC2 remains present as indicated by normal levels of SUZ12.

(D) Affinity purification of full-length epitope tagged wild-type (WT) KDM2B and KDM2BΔCxxC followed by tandem mass spectrometry-based analysis of associated proteins. The mascot score and percentage coverage is indicated for the KDM2B/PRC1 complex components. Importantly, removal of the ZF-CxxC exon generates a product that still associates with the PCGF1/PRC1 variant complex but lacks its capacity to bind nonmethylated DNA.