Identification and characterization of conserved noncoding cis-regulatory elements that impact Mecp2 expression and neurological functions

Abstract

While changes in MeCP2 dosage cause Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), its transcriptional regulation is poorly understood. Here, we identified six putative noncoding regulatory elements of Mecp2, two of which are conserved in humans. Upon deletion in mice and human iPSC-derived neurons, these elements altered RNA and protein levels in opposite directions and resulted in a subset of RTT- and MDS-like behavioral deficits in mice. Our discovery provides insight into transcriptional regulation of Mecp2/MECP2 and highlights genomic sites that could serve as diagnostic and therapeutic targets in RTT or MDS.

Keywords: MeCP2; cis-regulatory elements; neurological disorders; noncoding.

Figure 1.

Identification of putative cis-regulatory elements of Mecp2. (A) ATAC-seq reads mapped to the Mus musculus genome (mm9) are shown from nuclei isolated from embryonic, postnatal, and adult mouse brains (specifically E12.5, P6, and 8 wk of age). Exons are denoted by solid blue rectangles, while introns are between exons. The 3′ UTR is represented by a thinner but longer blue block relative to the coding exons. Note that locus is transcribed in the opposite direction relative to the image shown (i.e., the promoter is at the right, and the 3′ UTR is at the left). Peaks 1, 2, 3, 5, and 6 were pursued for functional analysis of cis-regulatory elements, whereas Peak-4 (which spanned the proximal promoter) was not studied further. (B) RT-qPCR shows Mecp2 mRNA expression in different knockout lines (n = 5). (C) Western blot analysis of mouse brain tissue shows MeCP2 protein expression levels in each different knockout line. Representative Western blot image of MeCP2 protein levels is shown with Gapdh as a loading control, and solid vertical lines indicate areas of the image that have been spliced to remove unneeded lanes (n = 4). All data were analyzed by one-way ANOVA followed by Dunnett post hoc test. Data are presented as mean ± SEM. (*) P < 0.05, (**) P < 0.01, (****) P < 0.0001.

Figure 2.

Peak-2^KO/y mice show age-related behavioral deficits and abnormal RTT-like gene expression. (A) Peak-2^KO/y mice traveled further and displayed more activity counts in the open field assay than their wild-type littermates in open field assay at 10 wk of age (n = 12). (B) Peak-2^KO/y mice show anxiety-like phenotypes as measured by decreased entries, less time spent, and decreased distance in the open arms in the elevated plus maze at 24 wk of age (n = 12). (C) Peak-2^KO/y mice show social deficits as measured by decreased time investigating a novel mouse in the three-chamber sociability assay at 24 wk of age (n = 12). (D) Peak-2^KO/y mice show social dominance deficits as measured by decreased winning percentage in the tube test at 40 wk of age (n = 12). Data are presented as mean ± SEM. All data were analyzed by two-tailed t-test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) not significant.

Figure 3.

Peak-6^KO/y mice show abnormal MDS-like gene expression and behavioral deficits. (A) Peak-6^KO/y mice show hypoactivity, with fewer activity counts in open field assay at 9 wk of age (n = 13–15). (B) Peak-6^KO/y mice show anxiety-like phenotypes as evidenced by decreased entries and time spent at the center in open field assay at 9 wk of age (n = 13–15). (C) Peak-6^KO/y mice show anxiety-like phenotypes as measured by reduced entries to the open arms in the elevated plus maze at 10 wk of age (n = 13–15). (D) Peak-6^KO/y mice show learning and memory deficits using a fear conditioning test at 36 wk of age (n = 13–15). Data are presented as mean ± SEM. All data were analyzed by two-tailed t-test. (*) P < 0.05, (**) P < 0.01.

Figure 4.

Deletion in conserved cis-regulatory elements of MECP2 affect the mRNA and protein levels in human iPSC-derived neurons. (A) MECP2 mRNA expression is reduced in Peak-2 KO and increased in Peak-6 KO iNeurons as measured by RT-qPCR (n = 5). (B) MeCP2 protein expression is reduced in Peak-2 KO and increased in Peak-6 KO iNeurons. Representative Western blot of MeCP2 levels in CRE deletion iNeurons with GAPDH displayed as loading control. Solid vertical line indicates a spliced region of the single gel image to remove unneeded lanes (n = 7). (C) Quantification of MeCP2 protein expression by Western blot normalized to GAPDH loading control (n = 7). All data were analyzed by two-tailed t-test. Data are presented as mean ± SEM. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

Figure 5.

Summary of Peak KO phenotypes and comparison with MeCP2 50% loss (Mecp2^flox/y) or MDS mouse model (MECP2-Tg1).