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Fig. 4. Dnmt1 represses activation of non-methylated transgenes in vivo and
xDnmt1 localises to non-methylated target genes. (A) N-terminal
xDnmt1 fusions bind dsDNA oligos. The domain structure of xDnmt1 (top).
xDnmt1-Gst fusions domains G1-G3 (black bars). CpGpos oligonucleotides were
used in pull-down assays with the three Gst fusions and Gst only. All three
constructs bound CpGpos compared with the Gst protein (G) lane. (B) In
vivo assays to test the repression activities of xDnmt1, hDNMT1WT and
hDNMT1C1226Y. Exogenous xSp1 was used to activate the Sp1-Luc
reporter in the presence of increasing amounts of Dnmt1 [black triangles;
0.125-2 µg plasmid DNA per transfection]. xSp1 activation of the reporter
alone was assigned 100%. Data were obtained from nine independent assays and
normalised to TK-Renilla. (C) Left panel: both forms of human DNMT1
(hDNMT1WT and hDNMT1C1226Y) are expressed equally after
transfection into 293T cells relative to PCNA and endogenous hDNMT1
(-plasmid). Right panel: N2A cells were transfected with T7xSp1 against low or
high xDnmt1 levels (black bar), cell extracts were blotted with 
-T7.
Tubulin was used as a loading control. (D) An xOct91
promoter (-111 to +343) reporter construct is repressed by co-transfection
with 500 ng of xDnmt1, hDNMT1WT and hDNMT1C1226Y but not by the
empty vector control. (E) Chromatin IP (ChIP) analysis shows
recruitment of GFP-xDnmt1 to the non-methylated xOct25 and
xCycD1 promoters, but not an -tubulin intron in A6 cells. Note
the enrichment of GFP-xDnmt1 at both promoters using -GFP (lane 2) but
not the control xDnmt1 lacking GFP (panel xDnmt1). Lane 1, input (1/20 used in
IP); -ve, no antibody control. (F) Bar chart shows fold enrichment of
GFP-xDnmt1 at the xOct25 (eightfold) and xCycD1 (4.5-fold)
promoters compared with a non-tagged control and with Tubulin.
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