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Fig. 4. Precocious astroglial differentiation is mediated by enhanced activation of
JAK/STAT signaling in Dnmt1–/– NPCs. (A)
Wild-type or a STAT-binding mutant form of the 1.9 kb rat GFAP
promoter-luciferase reporter constructs were co-transfected with the
renilla-TK control plasmid into 3 day cultured E11.5 control and
Dnmt1–/– CNS NPC cultures. After 24 hours,
cells were lysed and subjected to dual-luciferase assays (Promega).
*P<0.001 compared with the control group
(Dnmt1+/+) without LIF treatment. **P<0.01
compared with the group of Dnmt1–/– cells
without LIF treatment (ANOVA with Post-hoc tests). (B) EMSA assay using a 25
bp unmethylated probe containing the STAT-binding element within the GFAP
promoter with nuclear extracts from cultured control and
Dnmt1–/– E11.5 CNS cells as in A. The identity
of the DNA-protein complex (*) was characterized using anti-STAT1 and
anti-STAT3 supershift assays (arrows). (C) Left panel, bFGF expanded
(gliogenic) cortical progenitor cells were left untreated or treated with LIF
for 30 minutes and subjected to chromatin immunoprecipitation (ChIP) assay
with an antibody against STAT3 (Santa Cruz). A control antibody,
anti-ß-galactosidase, was used to control for ChIP assay specificity. In
the right two panels, ChIP assays were performed on 3-day-old cultured control
(con) and Dnmt1–/– (mut) E11.5 CNS cells using
the STAT1 and STAT3 antibodies. (D,E) E11.5 control
Dnmt1+/+ and Dnmt1–/– CNS
NPCs were cultured for 48 hours and co-transfected with a
ß-gal-expressing construct and a control plasmid (con) or a
dominant-negative STAT3F plasmid (STAT3F). After another 48 hours, cells were
fixed and double-stained with antibodies against GFAP (green) and ß-gal
(red), and counted for the percentage (mean±s.e.m.) of GFAP and
ß-gal double-positive cells over total ß-gal positive cells.
*P<0.01 compared with the Dnmt1+/+ (con)
group. **P<0.01 compared with the group of
Dnmt1–/– cells with ß-gal transfection
(con) (ANOVA with Post-hoc tests).
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