Identification of cis-Regulatory Elements for MECP2 Expression
Purpose: Rett syndrome (RTT) is an X-linked dominant disabling neurodevelopmental disorder caused by loss of function mutations in the MECP2 gene, located at Xq28, that encodes a multifunctional protein. While 70-80% of RTT females have de novo coding region mutations and ~ 10% have large deletions, other cases could result from mutations in non-coding regions that affect regulatory elements. MECP2 expression is regulated in a developmental and cell-type specific manner. The need for tightly controlled MeCP2 levels in brain is strongly suggested by neurologically abnormal phenotypes of mouse models with mild overexpression and mental retardation in human males with MECP2 duplication. We set out to identify long-range cis-regulatory sequences that differentially regulate MECP2 transcription, and when mutated, may contribute to the pathogenesis of RTT or X-linked mental retardation.
Methods and Results: By inter-species sequence comparison using bioinformatics tools, we detected 27 highly conserved non-coding DNA sequences within a 210 kb region covering MECP2. By performing luciferase reporter assays in four different human cell lines, we functionally confirmed four enhancer and two silencer elements. The capability of the identified regulatory elements to bind transcription factors or other proteins was tested by gel shift assays. We dissected the human MECP2 promoter region by reporter assays with deletion constructs and located the core promoter. By using chromatin conformation capture (3C) assays, we were able to document long rang interactions of three enhancers and two silencers with the MECP2 promoter. Acting over distances of up to 130 kb, these elements are likely to affect the specific regulation of MECP2 transcription.
Conclusions: Our study has defined the “MECP2 functional expression module”. Further research will be focused on validating the neuronal-specific role of each cis-acting element by quantitative 3C assay and transgenic mouse models.