From Function to Phenotype: Impaired DNA Binding and Clustering Correlates with Clinical Severity in Males with Missense Mutations in MECP2

Abstract

Mutations in the MECP2 gene cause Rett syndrome (RTT). MeCP2 binds to chromocentric DNA through its methyl CpG-binding domain (MBD) to regulate gene expression. In heterozygous females the variable phenotypic severity is modulated by non-random X-inactivation, thus making genotype-phenotype comparisons unreliable. However, genotype-phenotype correlations in males with hemizygousMECP2 mutations can provide more accurate insights in to the true biological effect of specific mutations. Here, we compared chromatin organization and binding dynamics for twelve MeCP2 missense mutations (including two novel and the five most common MBD missense RTT mutations) and identifiedacorrelation with phenotype in hemizygous males. We observed impaired interaction of MeCP2-DNA for mutations around the MBD-DNA binding interface, and defective chromatin clustering for distal MBD mutations. Furthermore, binding and mobility dynamics show a gradient of impairment depending on the amino acid properties and tertiary structure within the MBD. Interestingly, a wide range of phenotypic/clinical severity, ranging from neonatal encephalopathy to mild psychiatric abnormalities were observed and all are consistent with our functional/molecular results. Overall, clinical severity showed a direct correlation with the functional impairment of MeCP2. These mechanistic and phenotypic correlations of MeCP2 mutations will enable improved and individualized diagnostics, and may lead to personalized therapeutic interventions.

The role of N-terminal modification of MeCP2 in the pathophysiology of Rett syndrome

“…No significant difference in the protein-chromocenter localization was noted, indicating that mislocalization of protein is unlikely to be involved in the etiopathogenesis of p.Ala2Val. Additionally, there were no significant differences in the average number and size of chromocenters in cells transfected with the WT and mutant MeCP2_E1 constructs, which rules out an effect of the mutation on the clustering and overall chromatin organization (33).…”

MeCP2 AT-Hook1 mutations in patients with intellectual disability and/or schizophrenia disrupt DNA binding and chromatin compaction in vitro

“…Both the variable severity of the clinical phenotype and functional studies have shown that MECP2 mutations do not always result in the complete loss of protein function. We have recently shown that variation in the clinical severity and diminished functionality for mutations at specific MeCP2 amino acid residues is related to the physical and chemical properties of the substituting amino acid (Sheikh et al., ). Here we found that the p.Arg190His mutation (the substitution of one positively charged amino acid with histidine, which is largely uncharged at pH 7.4 but still retains some ability to bind the DNA) within the AT‐Hook1 domain does not result in the complete loss of MECP2‐DNA binding, whereas loss of DNA compaction was observed in vitro (Figure C).…”

“…These results, obtained using an AT‐rich double stranded DNA substrate, were corroborated using DNA from the BDNF ‐148 bp promoter sequence (Figure A–C), with the binding rate differing for p.Arg190, p.His190, and p.Cys190 (Figure D). Previously we reported that several residues from the distal part of MBD and ID (p.Asn126, p.Ala140, p.Pro152, p.Arg167) may contribute either to the structural integrity of the MECP2 protein, or to the binding of other nuclear proteins, which is crucial for the proper clustering of chromatin (Sheikh et al., ). Here, we propose another mechanism, that mutation within the AT‐hook1 site in MECP2 can also disrupt DNA compaction, suggesting that AT‐Hook1‐DNA binding is also crucial for chromatin clustering and correctly compacting chromatosomes to ensure proper chromosomal condensation (Figure C).…”

“…Cell line C2C12 (mouse skeletal muscle myoblast, ATCC, Manassas, VA) was used due to the relatively prominent chromocenters and low endogenous MECP2 expression (Agarwal et al., ; Sheikh et al., ) In order to express MECP2 in a mammalian system, the full‐length MECP2_E1 gene was amplified using reverse transcriptase PCR followed by cloning into the expression construct pcDNA3.1 CT‐GFP‐TOPO (placing GFP at the C‐terminal end of MECP2), according to the manufacturer's instructions (Life Technologies, Carlsbad, CA). Mutant (p.Arg190His and p.Arg190Cys) MECP2_E1‐GFP constructs were generated by site‐directed mutagenesis (as described above).…”

“…There are currently over 200 known mutations in MECP2 that cause RTT, with mutations detected in more than 90% of classical Rett patients, and almost 80% of those mutations have been found in exons 3 or 4 (Christodoulou, Grimm, Maher, & Bennetts, ; Krishnaraj, Ho, & Christodoulou, ). A wide range of phenotypic/clinical severity, ranging from neonatal encephalopathy to psychiatric disorders in hemizygous males shows that the functional effect of each mutation is strictly dependent on the location and nature of mutations with in the MECP2 protein (Sheikh et al., ). The first boy reported with a MECP2 mutation (NM_004992.3: c.473C > T; p.(Thr158Met)) had symptoms such as static encephalopathy, profound developmental delays, hypotonia, seizure, acquired microcephaly, constipation, and respiratory irregularities including central apnea (Villard et al., ).…”