4. CpG hypermethylation Data from literature RESULT: Genes that are hypermethylated at CpG islands in cancer cells (HCT- 116 cell line) are NOT highly methylated in ES or EC (Tera-1 and Tera-2) cells

5. EC cells retain a plasticity of expression that is lacking in adult cancers (a)RT-PCR of genes hyper-methylated in adult cancers (low # means higher gene expression); (b)SKIP (c)RT-PCR for RNA from Tera-2 cells grown as xenografts in NOD/SCID mice +/- ATRA

6. Promoter regions of studied genes contain active (dimethyl-H3K4) and repressive (trimethyl-H3K27) histone modifications in ES cells How detect methylated histone sites? Chromatin immunoprecipitation (ChIP) using antibodies to di- and tri- methylated histones; use beads to isolate antibody containing complexes Data in panel (a) PcG proteins - interact with methyltransferases and bind nucleosomes to compact chromatin; these proteins can lead to repression of genes, such as genes involved in body patterning and stem cell self-renewal (panel b)

7. Same experiment as in Fig 3a but with EC cells; see mostly same results EXCEPT that these cells have acquired two additional KEY repressive marker characteristics for adult cancer di- and tri-Methylated H3K9 Panels (b) and (c are quantification of data in gel Protein levels of repressive histone modifications in Tera2 (EC cells) vs. ES cells

8. Use EC cells and investigate what happens to chromatin findings when cells differentiate (ATRA is added as a differentiation agent) (a)Using Tera-2 cells and ChIP with antibodies to PcG proteins; key PcG proteins (SUZ12, EZH2, SirT1) were enriched at promoters of genes in EC cells (b) After ATRA treatments - protein levels mostly went down and (c protein localization to promoter went down (d) Quantitative ChIP after ATRA shows downregulated and upregulated genes

9. Overexpression of Bmi1 can cause promoter DNA methylation of SFRP5 gene in EC cells Bmi1 is a central protein component of PRC (polycomb repressive complex) and it recognizes methylated histones; endogenous in Tera2 cells (a) Overexpression after 10 cell passages (b) See increased cell proliferation (c methylation-specific PCR using bisulfite; bisulfite reacts with unMeth-DNA (C  U) but NOT meth-DNA, then primers specific to bisulfite modified DNA are used (d) ChIP to show methylated sites stay the same

10. CONCLUSIONS Genes showing DNA hypermethylation in adult cancers usually lack such DNA methylation in normal/neoplastic embryonic cells (FIRST table of data) BUT chromatin of genes is ~same in embryonic cancer cells and adult cancer cells (this paper’s results)