Supplementary Methods
Plasmids constructs. We cloned EZH2 and DNMT3B fragments into the vector pGEX
(Pharmacia) by PCR using appropriate sets of primers. GST-EZH2 was expressed from
pGEX-EZH2, a kind gift of A. Otte. GST-SUV39H1 and GST-DNMT1 as well as GSTDNMT3A were previously described.
GST pull-down and DNA methyltransferase assays. Recombinant proteins were expressed
in and purified from E. coli Top10 or Bl21. DNMT1, DNMT3A, and DNMT3B were in vitro
translated
respectively
from
pcDNA3-Myc-DNMT1,
pcDNA3-Myc-DNMT3A,
and
pcDNA3-Myc-DNMT3B. In vitro DNA methyltransferase assays were performed after
incubating HeLa nuclear extracts with immobilized GST-EZH2, GST-SUV39H1 or GSTPLZF (a kind gift of C. Beaudouin). For immunoprecipitations preceding the DNA
methyltransferase assay, we used pre-immune serum, anti-EZH2 and anti-PLZF (F15, Santa
Cruz) antibodies. The results are averages of at least three independent experiments with error
bars displaying standard deviations (s.d). In GST pull-downs experiments, input 10%,
corresponds to 10% of the IVT product used in the reaction.
Antibody generation. Antibodies against a synthetic peptide encompassing residues 14-30
(14-CWRKRVKSEYMRLRQLK-30) of human EZH2 and coupled to keyhole limpet
haemocyanin were raised in a rabbit and affinity-purified on the peptide coupled to bovine
serum albumin and linked to CNBr-activated Sepharose 4B (Amersham Biosciences).
Immunoprecipitations and western blot analysis. HeLa nuclear extracts (Cilbiotech) were
incubated at 4°C overnight with antibodes in IPH buffer. Antibodies used were anti-DNMT1
(ab13537, Abcam), anti-DNMT3A (IMG268, Imgenex), anti-DNMT3B (IMG184, Imgenex),
anti-EZH2 and anti-EED (07-368, Upstate). For control western blotting of RNAi-treated
cells, we used antibodies against EZH2, DNMT1 (a gift of S. Pradhan), DNMT3A, DNMT3B
(gifts of E. Li), and Actin (A5316, Sigma).
5-aza-dC treatment . U2OS cells were treated with 5-aza-deoxycytidine (1 µM final; Sigma)
3 days with drug refreshment every 24 hours. 5-aza-dC had no effect on EZH2 expression
levels (data not shown).
RNA interference and retroviral infection. The target sequence used for gene silencing was
inserted into the pRetroSuper (pRS) retroviral vector according to the manufacturer’s
recommendations (OligoEngine). Primer sequences are available on request. Retrovirus
production by 293 GP cells and infection of target cells were performed. Infected cells were
selected with 1 µg/ml puromycin (Sigma).
Chromatin
Immunoprecipitation
(ChIP).
Chromatin
from
U2OS
cells
was
immunoprecipitated with antibodies against EZH2, H3K27me3 (07-449, Upstate), DNMT1
(ab5208, Abcam), DNMT3A (IMG268, Imgenex), DNMT3B (ab2851, Abcam), RNA
polymerase II (sc-899, Santa Cruz) and also an IgG control (sc-2027, Santa Cruz). Previously
described specific primers16 were used to detect human MYT1, WNT1, KCNA1 or CNR1 by
PCR in immunoprecipitated DNA. The number of cycles and the amount of template were
varied to ensure that results were within the PCR linear range.