|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 5. Changes of DNA methylation on the GFAP and STAT1 promoters when NPCs become
gliogenic in control and Dnmt1–/– cells. (A)
Bisulfite sequencing analysis on eight CpG sites surrounding the STAT1/3
binding elements within the mouse Gfap promoter. The percentage of
methylation at each of the 8 CpG sites was plotted. (B) Bisulfite sequencing
analysis shows selective demethylation occurs at the –499 CpG site but
not at the –594 CpG site during 24-96 hours of culturing period of
wild-type E11.5 cortical cells. (C) Methylation-specific SNuPE assay was used
to independently quantify the extent of methylation at the single CpG site
lying within the STAT binding element in 1- and 4-day-old cultured E12.5
control (con) and Dnmt1–/– (mut) CNS cells and
in E18.5 brain samples in vivo. (D) Bisulfite sequencing analysis of eight CpG
sites within the Stat1 promoter (between –731 bp and –409
bp promoter region of the gene) in E18 control and
Dnmt1–/– CNS samples. (E,F). E11 control (con)
or Dnmt1–/– (mut) NPCs were transfected with
either a ß-gal expression vector or a CAG-promoter-Dnmt1 expression
plasmid (Chen et al., 2003)
within the first 24 hours of cell culturing. After an additional 4 days of
culturing in the presence of LIF (50 ng/ml) to promote glial differentiation,
cells were double-labeled with GFAP/ß-gal or GFAP/Dnmt1 (E) and
quantified for the percentage of GFAP+/ß-gal+ or GFAP+/Dnmt1+ cells as
plotted in F. Dnmt1 overexpression cells can be easily detected by the strong
Dnmt1 staining signals (arrows). Two arrowheads in the control culture
indicate the typical nuclear staining pattern of the endogenous Dnmt1 protein.
*P<0.001 compared with control (Dnmt1+/+) with
ß-gal plasmid transfection. **P<0.001 compared with
Dnmt1–/– cells with ß-gal plasmid
transfection (ANOVA with Post-hoc tests).
|
