Abstract
SummaryIn mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.
Highlights
Methylation occurs at the cytosine base of CpG dinucleotides, to form 5-methylcytosine (5mC), which is sometimes called ‘‘the fifth nucleotide of DNA’’ based on its heritability. 5mC is frequently concentrated in repetitive sequences, such as pericentromeric regions and the transposable elements of normal somatic cells (Yamagata et al, 2007; Yoder et al, 1997), and is enriched in specific gene loci, such as differentially methylated regions of imprinted genes (Bird, 2002)
DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization
We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status
Summary
Methylation occurs at the cytosine base of CpG dinucleotides, to form 5-methylcytosine (5mC), which is sometimes called ‘‘the fifth nucleotide of DNA’’ based on its heritability. 5mC is frequently concentrated in repetitive sequences, such as pericentromeric regions and the transposable elements of normal somatic cells (Yamagata et al, 2007; Yoder et al, 1997), and is enriched in specific gene loci, such as differentially methylated regions of imprinted genes (Bird, 2002). Hypermethylation of CpG islands and hypomethylation of repeated DNA elements are key features of cancers (Bergman and Cedar, 2013; Ehrlich, 2002, 2009) Once these CpG sites are methylated, they are recognized by proteins of the methyl-CpG-binding domain (MBD) family, which recruit additional protein complexes to these regions to repress gene expression and/or to generate a higher-order condensed chromatin structure called heterochromatin, to stabilize chromatin organizations (Bird, 2002; Clouaire and Stancheva, 2008). The discovery of proteins of the ten-eleven translocation (TET) family as 5mC hydroxylases further supports this notion that DNA methylation is dynamic (Guo et al, 2011; Ito et al, 2010; Wu and Zhang, 2010)
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