Abstract
A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.
Highlights
Epigenetics is the study of mitotically and/or meiotically stable but reversible modifications to nucleotides or higher order chromatin structure that can alter expression patterns of genes in the absence of changes to the underlying DNA sequence [1]. These modifications occur on multiple levels, such as 5-methyl-cytosine (5-meC) DNA methylation, post-translational modifications of histones bound by protein domains that serve as epigenetic writers, readers and erasers and noncoding RNAs that assist in the recruitment of chromatin modifying proteins to DNA [2]
We demonstrate for one such epigenetic barrier in a specific gene context, that removal of CGI methylation from the promoter of the X-chromosomal gene CDKL5 by directing a fusion of the catalytic domain of ten-eleven translocation dioxygenase 1 (TET1) to dCas9 results in reactivation of gene expression in a targeted manner
We demonstrate that programmable transcription using a transactivator achieves a moderate but significant CDKL5 upregulation when compared to other reported CRISPRa target sites [52] that was achieved across several cell lines
Summary
Epigenetics is the study of mitotically and/or meiotically stable but reversible modifications to nucleotides or higher order chromatin structure that can alter expression patterns of genes in the absence of changes to the underlying DNA sequence [1] These modifications occur on multiple levels, such as 5-methyl-cytosine (5-meC) DNA methylation, post-translational modifications of histones bound by protein domains that serve as epigenetic writers, readers and erasers and noncoding RNAs that assist in the recruitment of chromatin modifying proteins to DNA [2]. One X chromosome randomly becomes inactive and is cytologically manifested during interphase as a perinuclear heterochromatic Barr body, which is clonally maintained through mitosis [7,8] This mechanism is mediated by the long noncoding RNA X-inactive specific transcript (XIST) expressed from the inactive X chromosome in cis [9], which serves as a guiding factor to tether Polycomb proteins for gene silencing to target sites on the X-chromatin [10]. XIST induces the formation of repressive heterochromatin through histone deacetylation [11], DNA methylation of CpG-island (CGI) promoters [12], di- and trimethylation of histone 3 at lysine 9 (H3K9me2/3) [13], the deposition and spreading of H3K27me across the inactive X-chromatin [14] and the H2A histone variant macroH2A [15]
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