Cell-type-specific repression by methyl-CpG-binding protein 2 is biased toward long genes

Abstract

Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome and related autism spectrum disorders (Amir et al., 1999). MeCP2 is believed to be required for proper regulation of brain gene expression, but prior microarray studies in Mecp2 knock-out mice using brain tissue homogenates have revealed only subtle changes in gene expression (Tudor et al., 2002; Nuber et al., 2005; Jordan et al., 2007; Chahrour et al., 2008). Here, by profiling discrete subtypes of neurons we uncovered more dramatic effects of MeCP2 on gene expression, overcoming the "dilution problem" associated with assaying homogenates of complex tissues. The results reveal misregulation of genes involved in neuronal connectivity and communication. Importantly, genes upregulated following loss of MeCP2 are biased toward longer genes but this is not true for downregulated genes, suggesting MeCP2 may selectively repress long genes. Because genes involved in neuronal connectivity and communication, such as cell adhesion and cell-cell signaling genes, are enriched among longer genes, their misregulation following loss of MeCP2 suggests a possible etiology for altered circuit function in Rett syndrome.

Keywords: MeCP2; Rett syndrome; cell adhesion; microarray.

Figure 1.

A, Schematic of cell-type-specific expression profiling. M1, Primary motor cortex. B, Genes dysregulated in Mecp2 KO mice. Log2 transformed fold-change relative to WT mean is shown for each KO sample. Columns correspond to samples and rows correspond to genes. Red and blue indicate upregulation and downregulation in KO respectively. Genes are first sorted by whether it is upregulated in all cell types affected, mixed (upregulated in some but downregulated in others) or downregulated in all cell types affected, then by the number of affected cell types. C, Venn diagram showing the number of genes affected. D, Magnification of B where ≥3 groups are affected.

Figure 2.

qPCR confirmation. A, Log2 fold-changes (WT/KO) calculated from microarray (x-axis) versus qPCR experiments (y-axis). Values from individual qPCR experiments are plotted against averaged microarray data (across 3 samples per cell type). Black circles, qPCR data from amplified cDNA (cDNA2); gray circles, unamplified cDNA (cDNA1). B, Similar plot to A but unamplified cDNA from tissue homogenates from BSt and Ctx were used for qPCR instead of sorted samples. Dotted lines indicate linear fits. For Ctx samples, a subset of genes affected in L5 were assayed and compared with L5 array data. For BSt samples, a subset of genes affected in LC were assayed and compared with LC array data.

Figure 3.

GO over-representation. For each GO Slim category and cell group, we calculated the degree of over-representation among MeCP2 affected genes. The odds ratio (actual number over expected number) and p values (from hypergemetric distribution, represented as −log10 transformed values) are plotted using grayscales. GO Slim categories are sorted by over-representation index (right column; see text). Top, Biological processes; bottom, cellular components.

Figure 4.

Gene length dependence of MeCP2 affected genes. A, Percentage of genes in GO cell adhesion category in bins of 500 genes sorted by length is shown against mean length for each bin. Entire genes are used as the population. B, Similar to A but for GO category RNA metabolism. C, Similar to A and B, but percentage of MeCP2 affected genes (from all conditions) are plotted. D, Average gene length of MeCP2 misregulated genes for upregulated genes (dark bars) and downregulated genes (white bars) are shown. Dashed line indicates population average (48 kbp). E, F Normalized average log fold-change in bins of 500 genes sorted by length is plotted against average gene length in each bin. For each plot, average of the left half of the plot is used as the normalization factor. These normalized plots are shown separately for downregulated genes (E) and upregulated genes (F).