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. 2019 Jul 3;5(7):eaax2887. doi: 10.1126/sciadv.aax2887

Fig. 2. A conserved region in CATACOMB is responsible for H3K27me2/3 global reduction.

Fig. 2

(A) Schematic representation of the CATACOMB gene locus with PhyloP-generated scores in hg19 genome assembly. Negative scores are represented by red tracks for predicted acceleration, and positive scores are represented by blue tracks, for predicted conservation in 100 vertebrates (Vert.) (species list can be found in the University of California Santa Cruz genome browser). Top: The green box indicates the highly conserved region named CONS. Bottom: A zoom-in of the CONS region at DNA and amino acid levels. The red box indicates the four amino acid residues, including M406, that resemble the sequence surrounding the H3K27M mutant histone, which is shown below for comparison. (B) Western blotting of total cell extracts from SV40 immortalized hEnSC WT (−) expressing CATACOMB, CATACOMB without the CONS domain (ΔCONS), and CATACOMB containing a methionine-to-lysine mutation in position 406 (M406K). Antibodies used for Western blotting are indicated in the panel. H3 served as a loading control. (C) Western blotting of total cell extract from 293T cells (−) or 293T cells expressing CATACOMB or the CATACOMB M406K mutant. Antibodies used for Western blotting are indicated in the panel. H3 served as a loading control. (D) FLAG-IP in 293T cells that are WT or express FLAG-HA EZH2. In both cells lines, we expressed untagged CATACOMB, CATACOMBΔ CONS, or CATACOMB M406K. Ten percent of the input and 50% of the IP material were loaded for Western blot analysis. Antibodies used for Western blotting are indicated in the panel. (E) FLAG-IP in mESCs that are either WT or Ezh2ΔSET. In both cells lines, we expressed FLAG-HA CATACOMB. Cells with no FLAG-HA CATACOMB expression were used as negative control (−). Five percent of the input and 30% of the IP material were loaded for Western blot analysis. Antibodies used for Western blotting are indicated in the panel. (F) Western blotting of chromatin extracts from cells described in (E). Antibodies used for Western blotting are indicated in the panel. H3 served as a loading control.