Transcript Document

CpG
hypermethylation
Data from literature
RESULT:
Genes that are
hypermethylated at
CpG islands in
cancer cells (HCT116 cell line) are
NOT highly
methylated in ES or
EC (Tera-1 and
Tera-2) cells
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
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)
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
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
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
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)