Inhibitors of differentiation (ID1, ID2, ID3 and ID4) genes are neuronal targets of MeCP2 that are elevated in Rett syndrome

Abstract

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder caused by mutations in MECP2, encoding methyl-CpG-binding protein 2. MeCP2 is a transcriptional repressor elevated in mature neurons and is predicted to be required for neuronal maturation by regulating multiple target genes. Identifying primary gene targets in either Mecp2-deficient mice or human RTT brain has proven to be difficult, perhaps because of the transient requirement for MeCP2 during neuronal maturation. In order to experimentally control the timing of MeCP2 expression and deficiency during neuronal maturation, human SH-SY5Y cells undergoing mature neuronal differentiation were transfected with methylated MeCP2 oligonucleotide decoy to disrupt the binding of MeCP2 to endogenous targets. Genome-wide expression microarray analysis identified all four known members of the inhibitors of differentiation or inhibitors of DNA-binding (ID1, ID2, ID3 and ID4) subfamily of helix-loop-helix genes as novel neuronal targets of MeCP2. Chromatin immunoprecipitation analysis confirmed binding of MeCP2 near or within the promoters of ID1, ID2 and ID3, and quantitative RT-PCR confirmed increased expression of all four Id genes in Mecp2-deficient mouse brain. All four ID proteins were significantly increased in Mecp2-deficient mouse and human RTT brain using immunofluorescence and laser scanning cytometric analyses. Because of their involvement in cell differentiation and neural development, ID genes are ideal primary targets for MeCP2 regulation of neuronal maturation that may explain the molecular pathogenesis of RTT.

Figure 1. Quantitative RT-PCR results for ID1-4 in SH-SY5Y, Mecp2^−/y mouse brain cDNA and NeuroD1, a downstream target of ID genes

A, B and C: Graphical representation of all four ID genes qPCR data reflect fold changes relative to the control (set to 1.0, indicated by hatched bar) following normalization of the data to GAPDH house keeping control using the comparative CT method. A) The qPCR data for ID1, ID2 and ID3 genes show a decreased expression with differentiation, in untransfected SH-SY5Y cells (D-UT) and increased expression with MeCP2 decoy (D-MD). qPCR data of ID4 shows an increased expression level following SH-SY5Y differentiation. ID4 was further increased in the D-MD transfected cells compared to 48 h untransfected (D-UT). Results shown represent mean ± SEM of three replicate experiments. B) The qRT-PCR was conducted in cDNA samples from Mecp2^−/yand Mecp2^+/ymice brains. The relative fold change differences for all four ID genes were higher in the Mecp2^−/y compared to Mecp2^+/yat P28 time point, and reached significance by t-test for Id3 and Id4 (P < 0.05). No increase in cDNA was observed at later time points (P49 and P70). C) The qRT-PCR results on a downstream target of ID genes, NeuroD1, showed lower expression in all three postnatal time points (P28, P49 and P70) in Mecp2^−/y brain compared to the Mecp2^+/y control brain, with significantly reduced expression at P49 and P70 by t-test (P ≤ 0.05). Each time point in bar graphs B and C represents mean ± SEM of three replicate experiments from two pairs of mice per time point.

Figure 2. Increased ID protein expression changes in Mecp2-deficient mice model detected by immunofluorescence and LSC

Immunofluorescence followed by analysis on LSC was performed on postnatal day 28, 5 μm sagittal brain sections of three pairs of Mecp2^−/y and Mecp2^+/y mice using antibodies recognizing ID1, ID2, ID3 and ID4. Representative LSC image showing protein expression differences in multiple regions of the brain of Mecp2^−/y mice compared to Mecp2^+/y controls for all four ID proteins. Each pixel represents individual nuclei colored blue (negative), green (low) or red (high) based on max pixel fluorescence histograms. For each section, the hemotoxylin and eosin (H&E) stained parallel slide was used to determine the tissue localization of cells and gating of individual brain regions for quantitative data shown in Table 3.

Figure 3. Increased ID protein expression changes in RTT cerebrum detected by immunofluorescence and LSC

Immunofluorescence using antibodies recognizing ID1-4 was performed on postmortem cerebral cortex samples from one male and two female RTT samples with and without MECP2 coding mutations (filled, grey histograms) and age-matched controls (open, black histogram) arranged on a tissue microarray. Expression of all four ID proteins was significantly increased in RTT brain samples compared to the controls. In contrast, histone H1 expression was not significantly changed (data not shown). Significance was determined using chi-square from two replicate experiments and the P values are denoted by asterix on the top right corner of each histogram. (* P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005).

Figure 4. Chromatin Immunoprecipitation (ChIP) analysis of MeCP2 binding to ID1, ID2 and ID3 promoters

Representative gel of PCR products using primers specific to each of the ID gene promoter following ChIP with C-terminal MeCP2 reactive antibody. The total DNA isolated from chromatin prior to IP (Input) was used as a positive control. The ChIP results show binding of MeCP2 near or within the promoter of ID1, ID2 and ID3 in differentiated SH-SY5Y cells (D-UT) and control decoy transfected cells (D-CD). For ID4, binding of MeCP2 was not observed near or within the promoter region assayed. Additionally, the binding of MeCP2 to SNURF/SNRPN promoter is shown as a positive control for ChIP assay conditions.

Figure 5. Model for role of ID proteins in regulating neuronal maturational differentiation

In immature neurons with high expression of ID proteins, heterodimers of bHLH-ID prevent DNA binding and expression of differentiation associated genes like NEUROD1. Upon neuronal maturation, elevated levels of MeCP2 repress the expression of ID genes, resulting in functional bHLH-bHLH dimers that can bind to E-box DNA sequences and activate transcription of differentiation associated genes such as NEUROD1, ASCL1 and NEUROG1.