Dual DNA Methylation Patterns in the CNS Reveal Developmentally Poised Chromatin and Monoallelic Expression of Critical Genes

Abstract

As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay). We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F1 hybrid clonal neural stem cell (NSC) lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1–2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

Random monoallelic expression: regulating gene expression one allele at a time

“…Rather than directly analyzing allele-specific expression of the transcripts themselves, alternative methodologies have been used to identify putative monoallelically expressed genes [30–32]. These approaches assume the active and inactive alleles are marked by different epigenetic features, such as DNA methylation [31,32] or histone modifications [30].…”

“…These approaches assume the active and inactive alleles are marked by different epigenetic features, such as DNA methylation [31,32] or histone modifications [30]. As these data-sets are derived from non-clonal cell populations to identify monoallelically expressed genes such analysis comes with several caveats.…”

“…Simultaneous DNA methylation and lack of DNA methylation was used to identify potential monoallelically expressed genes in the mouse CNS [31,32], of which ~12% were confirmed to be monoallelically expressed in clonal cell populations [32]. The remaining genes likely reflect false positives due to the heterogeneous mix of cell types used in the original assay.…”

Cubilin expression is monoallelic and epigenetically augmented via PPARs

“…This phenomenon, called monoallelic expression [18], can occur through chromosomal inactivation (e.g., X inactivation), autosomal gene imprinting or random gene inactivation [19]. Monoallelic expression of a variety of autosomal genes have been described [19,20], including p120 catenin [21], certain cytokines [22], olfactory receptors and antigen receptors [23,24]. Here we explored the possibility that cubilin (Cubn), an autosomal gene [25], is regulated through epigenetic mechanisms and whether such processes might have consequences on cubilin function and on the expression of its partners, amnionless and megalin.…”

The Tumor Suppressor Function of LGI1

“…The results of tiling array assays (MAUD assay) for the identification of methylated or unmethylated DNA sequences, which can accommodate the entire mouse brain transcriptome, revealed that LGI1 is one of the genes presenting dual methylation pattern of the regulatory regions determining monoallelic expression (Wang et al, 2010). Although mutations of tumor suppressor genes are usually inherited as recessive traits, because both alleles must be inactivated to eliminate gene function, in case of monoallelic expression heterozygous mutations of the ummethylated allele might be sufficient to abolish expression.…”