Abstract
Abnormal regulation of DNA methylation and its readers has been associated with a wide range of cellular dysfunction. Disruption of the normal function of DNA methylation readers contributes to cancer progression, neurodevelopmental disorders, autoimmune disease and other pathologies. One reader of DNA methylation known to be especially important is MeCP2. It acts a bridge and connects DNA methylation with histone modifications and regulates many gene targets contributing to various diseases; however, much remains unknown about how it contributes to cancer malignancy. We and others previously described novel MeCP2 post-translational regulation. We set out to test the hypothesis that MeCP2 would regulate novel genes linked with tumorigenesis and that MeCP2 is subject to additional post-translational regulation not previously identified. Herein we report novel genes bound and regulated by MeCP2 through MeCP2 ChIP-seq and RNA-seq analyses in two breast cancer cell lines representing different breast cancer subtypes. Through genomics analyses, we localize MeCP2 to novel gene targets and further define the full range of gene targets within breast cancer cell lines. We also further examine the scope of clinical and pre-clinical lysine deacetylase inhibitors (KDACi) that regulate MeCP2 post-translationally. Through proteomics analyses, we identify many additional novel acetylation sites, nine of which are mutated in Rett Syndrome. Our study provides important new insight into downstream targets of MeCP2 and provide the first comprehensive map of novel sites of acetylation associated with both pre-clinical and FDA-approved KDACi used in the clinic. This report examines a critical reader of DNA methylation and has important implications for understanding MeCP2 regulation in cancer models and identifying novel molecular targets associated with epigenetic therapies.
Highlights
Epigenetic dysregulation involving mutations or abnormal expression of DNA methylation readers has been associated with a broad spectrum of disorders that range from Rett Syndrome to human cancers [1,2,3,4,5,6,7], and alterations in both the writing and reading of epigenetic marks have been linked with tumor progression at every stage [8,9,10,11,12]
We first examined methyl-CpG-binding protein 2 (MeCP2) protein expression in breast cancer cells and noted a range of expression across all lines with higher expression [64, 65] in MDA-MB-468 and BT-549 cells (Figure 1A). Both of these lines are derived from triple negative breast cancer (TNBC) which lack the expression of hormone receptors (ER and PR) and do not overexpress the growth factor receptor, HER2
MeCP2 Chromatin Immunoprecipitation (ChIP)-Seq had not been done in MDA-MB-468 cells and our analysis revealed that MeCP2 binds to a wide spectrum of target genes (~20,000 in MDA-MB-468 and ~1,337 in MCF7 cells) ranging from miRNA, long non-coding short hairpin RNA (RNA) (lncRNA), snRNA, processed and unprocessed pseudogenes, antisense and protein-coding genes
Summary
Epigenetic dysregulation involving mutations or abnormal expression of DNA methylation readers has been associated with a broad spectrum of disorders that range from Rett Syndrome to human cancers [1,2,3,4,5,6,7], and alterations in both the writing and reading of epigenetic marks have been linked with tumor progression at every stage [8,9,10,11,12]. DNA methylation readers, such as methyl-CpG-binding protein 2 (MeCP2), bridge DNA methylation and histone modifications by binding to methylated DNA and recruiting co-repressor proteins [25,26,27,28] While both normal and abnormal DNA methylation is read by MeCP2, much remains unknown about its role and regulation in cancer-associated pathologies. We previously reported that inhibition of SIRT1 triggers acetylation of endogenous MeCP2 at lysine (K171), a site that regulates MeCP2 interaction with HDAC1 and ATRX [44] These findings demonstrated that MeCP2 post-translational modifications (PTMs) can critically impact its function, yet few PTMs have been mapped despite the potential that they might affect substrate specificity [35, 45]. This knowledge gap is especially important given reports demonstrating unique characteristics of MeCP2
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