Table 1.
Primer sequences.
Figure 1.
POMC gene expression and methylation changes in MBH of fetal alcohol exposed rat offspring.
A. POMC mRNA levels in MBH of AD, PF and AF rat offspring were measured by quantitative RT-PCR and the amounts were normalized with GAPDH values and expressed as relative mRNA levels. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, AF vs AD or PF. B. POMC promoter methylation levels in MBH of AD, PF and AF rat offspring were assayed by real time methylation specific PCR. POMC promoter methylation levels were measured as ratios of methylated verses unmethylated DNA values. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; ***, P<0.001, AF vs AD or PF.
Figure 2.
Changes in MeCP2 gene and protein levels in MBH of fetal alcohol exposed rat offspring.
A. MeCP2 mRNA levels in MBH of AD, PF and AF rat offspring. MeCP2 mRNA levels were measured by quantitative RT-PCR and the amounts were normalized with GAPDH values and expressed as relative mRNA levels. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, AF vs. AD or PF. B. A representative western blot gel demonstrating changes in MeCP2 protein levels (top) and actin levels (loading control, bottom) in MBH of AD, PF and AF rat offspring along with quantification measurements were represented as relative protein level (MeCP2/actin). Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; **, P<0.01, AF vs. AD or PF. C. MeCP2 protein levels in β-endorphin neurons in the ARC of AD, PF and AF rat offspring were measured by double immunofluorescence methods (MeCP2 proteins are shown in green and β-endorphin shown in red). Representative photographs show double-labeled cells in each group. Histograms show the mean ± SEM (n = 6) values of percent β-EP cells expressing MeCP2 and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; ***, P<0.001, AF vs. AD or PF.
Figure 3.
Changes in MeCP2 binding onto POMC promoter in ARC and PVN of fetal alcohol exposed rat offspring.
A. Schematic representation of POMC promoter with CpG sites and primers flanking MeCP2 binding site. B. The levels of MeCP2 binding on POMC promoter in ARC and PVN of AD, PF and AF rat offspring were measured by ChIP assay. MeCP2 bound DNA pulled by its specific antibody was amplified by real time PCR using primers specific for the POMC promoter. MeCP2 enriched DNA was normalized with GAPDH and expressed as fold enrichment (MeCP2/GAPDH). AD and AF are ARC samples of ad libitum-fed and alcohol-fed rats. ADP and AFP are PVN samples of ad libitum-fed and alcohol-fed rats. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, AF vs. AD; a, P<0.001, ADP and AFP versus AD or AF.
Figure 4.
Effects of lentiviral knockdown of MeCP2 on POMC gene expression levels in fetal alcohol exposed rat offspring.
A. A representative western blot gel showing MeCP2 protein levels (top) and actin levels (bottom) in MBH of scr sh (scr) or MeCP2 sh (MeCP) RNA-treated AD, PF and AF rat offspring. Histograms showing MeCP2 and actin ratio values in MBH of AD, PF and AF rats.treated with scr sh or MeCP2 sh RNA. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, MeCP2 sh vs. scr-sh; a, P<0.05, AF vs. AD or PF. B. POMC mRNA levels in MBH of scr-sh and MeCP2 sh RNA of AD, PF and AF rat offspring. POMC mRNA amounts were normalized with GAPDH and expressed as relative mRNA level. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, AF- vs. AD- or PF-scr-sh-treated groups; a, P<0.05, MeCP2 sh vs. scr-sh (AF group).
Figure 5.
Effect of MeCP2 knockdown on stress axis responsiveness in fetal alcohol exposed rat offspring.
A. CRH mRNA levels in the MBH of scr sh- or MeCP2 sh RNA-treated AD, PF and AF rat offspring. CRH mRNA amounts were normalized with GAPDH and expressed as relative mRNA level. Data are mean ± SEM (n = 6) and were analyzed using two-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, AF vs. AD or PF groups; a, P<0.05, MeCP2 sh vs. scr-sh (AF group). B. ACTH levels in plasma of AD, PF or AF rats treated with either scr sh or MeCP2-sh RNA at 0 h and 2 h after LPS treatment. Data are mean ± SEM (n = 6) and were analyzed using two-way ANOVA with Bonferroni post-hoc test; *, P<0.05, AF vs. AD or PF (within Scr sh treatment groups); a, P<0.05, MeCP2 sh vs. scr-sh (AF groups). C. Corticosterone levels in AD, PF or AF rats treated with either scr sh or MeCP2 sh RNA at 0 h and 2 h after LPS treatment. Data are mean ± SEM (n = 6) and were analyzed using two-way ANOVA with Bonferroni post-hoc test; *, P<0.05, AF vs. AD or PF (within Scr sh treatment groups); a, P<0.05, MeCP2 sh vs. scr-sh (AF groups).
Figure 6.
A Model illustrating the role of promoter methylation and MeCP2 binding in regulation of POMC expression following fetal alcohol exposure.
In control rat offspring transcription factor binds to hypomethylated POMC promoter and activates transcription (top). Fetal alcohol exposure promotes hypermethylation of the POMC promoter and recruits MeCP2. MeCP2 binding to CpG methylated POMC promoter prevents the transcription factor's ability to bindto activate gene transcription.