Fig 1.
Circadian oscillations of MeCP2 and CLOCK proteins in mouse frontal cortex.
RT-qPCR results and densitometric analysis of WB experiments, showing differences in (A) MeCP2 and (B) CLOCK expression. Mice were under constant 12 hour light-dark cycles (white and grey background represent lights on and off, respectively) and were processed at the indicated ZTs (Zeitgeber times in hours) (n = 10/ time point, means ± SEM are represented). Both MeCP2 and CLOCK levels are significantly different between ZTs 6 and 18. (C), (D) Representative MeCP2 and CLOCK WBs (white and black bars represent, respectively, lights on and off). *P<0.05, **P<0.005, ***p<0.0005 in two-tailed Student’s t-tests.
Fig 2.
Changes in chromatin accessibility at the Bdnf, Dlk and Sst promoters correlate with MeCP2 levels.
(A) MNase digestions, chromatin fractionation, and following RT-qPCRs revealed in all three cases a progression from a less accessible status at ZT 6 to a transcriptionally permissive status at ZT 18 (n = 3–5 mice/time point; graphs show mean ± SEM). (B) Correlation of promoter accessibility with MeCP2 levels. *P<0.05, **p<0.005 in two-tailed Student’s t-tests. Correlation p-values correspond to the linear regression coefficient.
Fig 3.
MeCP2-targets expression patterns also exhibit circadian changes.
(A) RT qPCR analysis demonstrates gene expression changes along light-dark cycles (n = 3–5 mice/time point; graphs show mean ± SEM). (B) Correlations between MeCP2-targets expression with MeCP2 protein levels as well as (C) with the accessibility to the corresponding promoters. *P<0.05, **p<0.005 in two-tailed Student’s t-tests. Correlation p-values correspond to the linear regression coefficient.
Fig 4.
Cartoon representation of the transitions observed during the mouse circadian cycle.
(A) The circadian variation of CLOCK and MeCP2 are accompanied by changes in the overall accessibility of brain chromatin to Mnase digestion. (B) Schematic representation of the MeCP2 changes observed during the circadian cycle. The overall increase in the MeCP2 level results in changes in its binding to the chromatin substrate, resulting in global changes in micrococcal nuclease accessibility (green scissors). (C) Additional MeCP2 binding to 5-hydroxymethylcytosines or 5-methylcytosines of certain chromatin domains and its possible association with different protein partners, results in the enhancement of its dual transcriptional activity, leading to either activation or repression of gene expression. In this representation, nucleosomes are in white, DNA is indicated as a black line, CpG dinucleotides are represented as lollipop structures black colored when methylated, grey when hydroximethylated and white when unmodified; MeCP2 partners are depicted as ovals or circles with question marks.