|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 1. Biochemical characterization of ICF mutations. (A) Schematic
diagram of the mouse Dnmt3b protein structure. The conserved PWWP and ATRX
domains, the methyltransferase motifs (I, IV, VI, IX and X), and the ICF
mutations that we introduced into mouse Dnmt3b cDNA are indicated.
The location of the interaction domain with Dnmt3a (see Fig. S1 in the
supplementary material) is also shown. (B) Interaction of ICF mutants
with wild-type Dnmt3a and Dnmt3b. COS-7 cells were transfected with two
expression vectors, one for myc-Dnmt3a (left panel) or myc-Dnmt3b1 (right
panel), and another for GFP-Dnmt3b1 or GFP-ICF mutants, as indicated.
GFP-tagged proteins were immunoprecipitated from cell extracts using an
anti-GFP antibody. Immunoblotting analysis of the immunoprecipitates was
carried out using anti-myc antibody (top panel). The middle and bottom panels
show the results of immunoblotting of the total cell extract (TCE) from
transfected cells with anti-myc and anti-GFP antibodies, respectively.
(C) Subcellular localization of Dnmt3b isoforms and ICF mutants.
GFP-tagged Dnmt3b isoforms or ICF mutants were expressed in NIH3T3 cells, and
the cells were fixed and analyzed by fluorescence microscopy. For each
construct, 200-300 transfected (green) cells were counted and the percentages
of cells showing different localization patterns are indicated. (D)
Stable expression of wild-type and mutant Dnmt3b in
Dnmt3a-/-, Dnmt3b-/- ES cells.
Expression vectors encoding mouse Dnmt3b1 (m3b1), A609T, D823G and PC (Dnmt3b
with its PC motif mutated) (top panel), and human DNMT3B (h3B1), A603T and
D817G (lower panel), were individually (or in a combination of two ICF
mutants) electroporated into late-passage 7aabb cells and selected in
blasticidin-containing medium. Blasticidin-resistant clones were analyzed by
immunoblotting using anti-Dnmt3b and anti-
-tubulin antibodies.
(E) DNA methylation analysis. Genomic DNA from the indicated ES cell
lines was digested with HpaII and analyzed by Southern hybridization
using a probe (pMO) for the endogenous C-type retrovirus repeats. DNA from
wild-type ES cells (J1) digested with MspI (M) was used as a control
for complete digestion.
|
