Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements

Abstract

MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs) within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum), whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute towards characterizing the expression profiles of Mecp2/MeCP2 isoforms and thereby provide insights on the potential role of MeCP2 isoforms in the developing and adult brain.

Figure 1. Transcript and protein expression of MeCP2 isoforms during mouse brain development.

(A) Western blot analysis of MeCP2E1 in the isolated nuclear extracts from the whole brain at the indicated developmental time points. ACTIN was used as a loading control. N = 3±SEM. (B) Quantitative RT-PCR with specific primers to detect Mecp2e1 and Mecp2e2 transcripts. N = 3±SEM. Significant differences are indicated with P<0.001***. (C) Similar to A for MeCP2E2. N = 3±SEM. For A and C, significant differences in the levels of each MeCP2 isoform between different developmental time points are found in Table S5. (D) Pearson's correlation analysis for indicated Mecp2 transcripts and MeCP2 protein levels. r =  Pearson's correlation coefficient, r² = coefficient of determination, P<0.05*. E: embryonic days, P: postnatal days, null: Mecp2 ^{tm1.1Bird y/−} brain tissue (nuclear extracts used as negative controls for A and C, total RNA was used as negative control in B).

Figure 2. Validation of anti-MeCP2E2 antibody and detection of MeCP2 isoforms in mouse brain.

(A) Schematic representation of MeCP2E1 and MeCP2E2 protein structures, differing only in their N-terminal sequences. MBD: methyl binding domain, ID: intervening domain, TRD: transcriptional repression domain, CTD: C-terminal domain (adapted from [21]). (B) Schematics of MECP2E1 (Retro-EF1α-E1) and MECP2E2 (Retro-EF1α-E2) retroviral vectors with C-MYC tag that were used for transfection of Phoenix cells (in C) and transduction of NIH3T3 cells (in D) (adapted from [18]). (C) Western blot (WB) experiment to detect MeCP2E2 expression in control non-transfected (NT), MECP2E1 transfected (E1-T), MECP2E2 transfected (E2-T), and E2-T pre-incubated with E2 antigenic peptide. Anti-MYC labelling was used as a positive control. (D) MeCP2E2 detection by immunofluorescence staining in transduced NIH3T3 cells with either a) MECP2E1, or b) MECP2E2 retroviral vectors. (E) Detection of endogenous MeCP2E2 by immunohistochemistry in the CA1 region of adult mouse hippocampus from a) wild type (WT) (Mecp2 ^{tm1.1Bird y/+}), and b) null (Mecp2 ^{tm1.1Bird y/−}) Mecp2 mice. (F) Controls to verify the specificity of anti-MeCP2E2 by IHC in the adult mouse brain; a) primary omission, b) anti-MeCP2E2 incubation with IgY, pre-incubation of the newly generated anti-MeCP2E2 antibody with the antigenic peptide against c) MeCP2E2, d) MeCP2E1, e) C-terminus of MeCP2. (G) Western blot to detect MeCP2E2 in the WT adult mouse brain and Mecp2 null mice. GAPDH was used as a loading control. (H) a) Confocal images of MeCP2E1 in WT adult mouse brain hippocampus CA1 region. b) Signal intensity profile analysis indicates the enrichment of MeCP2E1 at the DAPI-rich heterochromatin regions of nuclei. (I) a) Confocal images of MeCP2E2 in WT adult mouse brain hippocampus CA1 region. b) Signal intensity profile analysis of MeCP2E1 and DAPI co-localization indicating MeCP2E2 detection at the DAPI-rich heterochromatin regions of nuclei. Scale bars represent 20 µm in D–E, 10 µm in D, and 2 µm in H–I.

Figure 3. Detection of MeCP2E1 and MeCP2E2 in different cell types of the hippocampus (CA1 region) in adult male mouse brain.

Left panel (A–F): MeCP2E1 and, Right panel (A1–F1): MeCP2E2. Expression of MeCP2 isoforms was analyzed in neurons (NEUN⁺), astrocytes (GFAP⁺), and oligodendrocytes (CNPase⁺). (A–C) and (A1–C1) represent ×40 images. Scale bars represent 20 µm. (D–F) and (D1–F1) represent confocal images of single nuclei. Scale bars represent 2 µm. (A–A1) and (D–D1): Expression of MeCP2 isoforms in NEUN⁺ neurons. (B–B1) and (E–E1): Expression of MeCP2 isoforms in GFAP⁺ astrocytes. (C–C1) and (F–F1): Expression of MeCP2 isoforms in CNPase⁺ oligodendrocytes. All images were taken from hippocampus of adult male mouse brain.

Figure 4. Detection of MeCP2E1 and MeCP2E2 in different cell types of the hippocampus (CA1 region) in adult female mouse brain.

Left panel (A–F): MeCP2E1 and, Right panel (A1–F1): MeCP2E2. Expression of MeCP2 isoforms was analyzed in neurons (NEUN⁺), astrocytes (GFAP⁺), and oligodendrocytes (CNPase⁺). (A–C) and (A1–C1) represent ×40 images. Scale bars represent 20 µm. (D–F) and (D1–F1) represent confocal images of single nuclei. Scale bars represent 2 µm. (A–A1) and (D–D1): Expression of MeCP2 isoforms in NEUN⁺ neurons. (B–B1) and (E–E1): Expression of MeCP2 isoforms in GFAP⁺ astrocytes. (C–C1) and (F–F1): Expression of MeCP2 isoforms in CNPase⁺ oligodendrocytes. All images were taken from hippocampus of adult female mouse brain.

Figure 5. Immunohistochemical detection of MeCP2E1 and MeCP2E2 in the adult mouse brain regions.

Left panel (A–J): MeCP2E1, and right panel (A1–J1): MeCP2E2. Expression of MeCP2 isoforms was analyzed in seven indicated brain regions of the adult mouse brain. (A–A1) Tiled image of whole hippocampus, and specific regions of hippocampus, (B–B1) CA2, (C–C1) CA3, (D–D1) Dentate gyrus (DG); (E–E1) Olfactory bulb; (F–F1) Striatum; (G–G1) Cortex; (H–H1) Thalamus; (I–I1) Brain stem; (J–J1) Cerebellum. Scale bars represent 200 µm in (A–A1) and 20 µm in (B–J and B1–J1). (K) Detection of MeCP2E1 and MeCP2E2 in three layers of the cerebellum; molecular layer (ml), Purkinje cell layer (pcl) and granule cell layer (gcl). Scale bars represent 20 µm. (L–N) Colocalization of MeCP2E1 and MeCP2E2 in the nuclei in the three layers of the cerebellum; (L) molecular layer, (M) Purkinje cell layer and (N) granular cell layer. Scale bars represent 2 µm. (O) Absence of MeCP2E1-, and MeCP2E2-specific signals in three layers of the cerebellum of the Mecp2 ^{tm1.1Bird y/−} null mouse brain [molecular layer (ml), Purkinje cell layer (pcl) and granule cell layer (gcl)]. Scale bars represent 20 µm. (P–R) Absence of MeCP2E1/- and MeCP2E2-specific signals in the nuclei in the three layers of the cerebellum of the Mecp2 ^{tm1.1Bird y/−} null mouse brain; (P) molecular layer, (Q) Purkinje cell layer and (R) granular cell layer. Scale bars represent 2 µm.

Figure 6. Transcript and protein expression of MeCP2 isoforms in adult mouse brain regions.

Expression of Mecp2/MeCP2 isoforms was analyzed in the indicated brain regions of the adult male murine brain. (A) Western blot (WB) analysis of MeCP2E1 in brain regions. ACTIN was used as a loading control. N = 3±SEM. Whole WT Mecp2 and null Mecp2 adult brains were used as controls. (B) Similar to A for MeCP2E2. N = 3±SEM. (C) Quantitative RT-PCR to detect transcript levels of Mecp2 isoforms in brain regions. N = 3±SEM. Significant differences between the two isoforms are indicated with P<0.0001****, P<0.001***, P<0.01** or P<0.05*. For A–B, significant differences in the levels of each MeCP2 isoform between different brain regions are found in Table S2. (D) Pearson's correlation analysis between Mecp2 transcript and MeCP2 protein expression. r =  Pearson's correlation coefficient, r² = coefficient of determination, P<0.001*** or P<0.05*. (E) (a) Semi-quantitative representation of MeCP2E1 and MeCP2E2 levels in WT Mecp2 and null Mecp2 adult brain. N = 3±SEM. (b) Quantitative RT-PCR to detect transcript levels of Mecp2 isoforms in WT Mecp2 and null Mecp2 adult whole brains. N = 3±SEM. Significant differences between the two isoforms are indicated with P<0.0001****, P<0.001***. OB: olfactory bulb, STR: striatum, CTX: cortex, HIPP: hippocampus, THAL: thalamus, BS: brain stem, CERE: cerebellum.

Figure 7. Bisulfite pyrosequencing analysis of DNA methylation status at the Mecp2 regulatory elements in adult murine brain regions.

(A) Schematic representation of regions within the Mecp2 promoter (R1–R3) and intron 1 (R4–R6) (not drawn to scale) . The first ATG of exon 1 is marked +1. (B) The graphs represent the percentage methylation (% Meth) observed at the individual CpG sites within analyzed sequences in seven indicated brain regions of adult mouse brain. a-a′: olfactory bulb, b-b′: striatum, c-c′: cortex, d-d′: hippocampus, e-e′: thalamus, f-f′: brain stem and g-g′: cerebellum. N = 5±SEM. Blue shaded regions show statistically significant differences between brain regions. For detailed comparison of statistical analysis, see Table S3.

Figure 8. Correlation analysis between DNA methylation at the Mecp2 regulatory elements and the expression of Mecp2 isoforms in the adult mouse brain regions.

(A) Graph represents Pearson's correlation coefficient (r) for correlation between DNA methylation at the Mecp2 regulatory elements and Mecp2e1 (black), and Mecp2e2 (white) expression in seven adult mouse brain regions. Stars (*) indicate statistical significance P<0.05*. N = 3. (B) Table representing Pearson's correlation coefficient (r) for correlation between DNA methylation at the Mecp2 regulatory elements and expression of Mecp2e1 (black), and Mecp2e2 (white). Stars (*) indicate statistical significance P<0.05*. N = 3.