MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2

Abstract

Background: MeCP2-a chromatin-binding protein associated with Rett syndrome-has two main isoforms, MeCP2-E1 and MeCP2-E2, differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest that they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored.

Results: Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl-binding domain (MBD) to interact with DNA as well as influencing the turn-over rates, binding dynamics, response to neuronal depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes.

Conclusions: Our study supports the idea that Rett syndrome might arise from simultaneous impairment of cellular processes involving non-overlapping functions of MECP2 isoforms. For instance, MeCP2-E1 mutations might impact stimuli-dependent chromatin regulation, while MeCP2-E2 mutations could result in aberrant ribosomal expression. Overall, our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.

Keywords: Chromatin; Isoforms; MeCP2; Rett syndrome.

Fig. 1

Biophysical characterization of the MeCP2-E1 and E2 NTD-MBD domain interaction with DNA. a Schematic representation of the MeCP2-E1 and E2 isoforms depicting the unique NTD amino acid sequences and shared domains. b Fluorescence thermal denaturation curves for E1 and E2 NTD-MBD protein fragments in the presence of unmethylated and mCpG-dsDNA. Unfolding traces were fitted considering a two-state unfolding model. c Unfolding stability parameters obtained from thermal denaturations followed by intrinsic tryptophan fluorescence. d Calorimetric titrations of E1 and E2 NTD-MBD proteins interacting with dsDNA plots show the thermograms (thermal power as a function of time) and the binding isotherms (normalized heats as a function of the dsDNA/protein molar ratio). e Buffer-independent dsDNA binding parameters (K_d, dissociation constant; ΔG: Gibbs free energy of interaction; ΔH: enthalpy of interaction; −TΔS: entropic contribution of interaction; ΔC_P: heat capacity of interaction; n_H: number of protons exchanged upon complex formation) obtained from calorimetric titrations at pH 7

Fig. 2

Isoforms N-terminal processing, turn-over rates, and dynamics. a Mass spectrometry sequencing of the N-terminal end of the MeCP2 protein (in vitro). N-terminal peptide coverage alignment chart and high-resolution mass spectra showing N-methionine excision (NME) and N-acetylation (NA) of the N-termini of MeCP2-E1 and MeCP2-E2. NA (+42 Da) of N-terminus amino acid is shown highlighted in yellow. b–d Cycloheximide-chase assays of the E1 and E2 MeCP2 isoforms. Densitometries and representative western blots performed after cycloheximide treatments of b SH-SY5Y cells overexpressing (OE) E1 and E2 isoforms fused to GFP, c differentiated SH-SY5Y, and d rat cultured cortical neurons with detection of endogenous E1 and E2 isoforms. e Densitometric analysis and representative western blots showing endogenous E1 and E2 levels in frontal cortices of mice euthanized at 12 a.m. and 12 p.m. f KCL depolarization and representative Western blots analysis of total endogenous MeCP2 of cultured cortical neurons and E1 and E2 of transfected cultured cortical neurons overexpressing Flag-MeCP2-E1 or E2. Represented data are mean ± S.E.M. (n = 7–8). * P < 0.05 two-tailed Mann–Whitney test. MeCP2 levels were normalized using β actin and/or histone H3

Fig. 3

Genome-wide distribution and dynamics of MeCP2 isoforms. a Bar plot depicting the distributions of regions enriched in the MeCP2 isoforms across eight defined genomic features. b DNA-binding motifs enriched for MeCP2-E1 (and excluding E2) shown as motif logos based on aligned, over-represented patterns found in the binding sites. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of symbols within the stack reflects the relative frequency of the corresponding amino or nucleic acid at that position. c ChIP-seq average profiles across 3 Kb upstream the TSS and 3 Kb downstream the TES of genic regions occupied by E1 at 12 a.m. and 12 p.m. (top) and E2 at 12 a.m. and 12 p.m. (bottom). d Details of the previous representations focusing in a 6 Kb region surrounding the TSS of E1 and E2 bound genes (top and bottom panels, respectively)

Fig. 4

MeCP2-E1 and E2 isoforms display diurnal dynamic genomic binding. a Heatmaps representing the log2 ratios obtained for E1 and E2 ChIP experiments; each column is divided into five clusters using the k-means algorithm. Protein occupancy is represented by color intensity, where the darker the color, the higher the protein enrichment. b Comparison of E1 enrichment at 12 a.m. vs. 12 p.m. showing occupancy differences in different clusters of interest. c Heatmap depicting the E2 12 a.m. vs. 12 p.m. shows a dynamic binding in clusters 4 and 5 (yellow and orange, respectively). d Left graphs: top-enriched functional pathways (−log10 (P)) of genes included in dynamic E1 and E2 gene clusters [Kyoto encyclopedia of genes and genomes (KEGG)]. Right graphs: ChIP-qPCR results validating MeCP2-E1 and E2 variations in occupancy of genes included in each of the gene clusters obtained

Fig. 5

MeCP2-E1 and E2 interacting proteins. a Schematic workflow of the proteomic analysis. b, c Proteins partners identified for each isoform. d Selected pathways enriched in E1 and E2 interacting proteins as identified by functional clustering (DAVID Gene Ontology Bioinformatics Resources). Newly identified interactors are shown in black and previously identified interactors are highlighted in orange