Abstract
Durable gene silencing through the formation of compact heterochromatin domains plays a critical role during mammalian development in establishing defined tissues capable of retaining cellular identity. Hallmarks of heterochromatin gene repression are the binding of heterochromatin protein 1 (HP1), trimethylation of lysine 9 on histone H3 (H3K9me3) and the methylation of cytosine residues of DNA. HP1 binds directly to the H3K9me3 histone modification, and while DNA methyltransferases have been found in complex with histone methyltransferases and HP1, there remains much to be known about the relationship between DNA sequence and HP1 in differentiated mammalian cells. To further explore this interplay in a controlled system, we designed a system to test the effect of promoter CpG content on the formation kinetics and memory of an HP1-mediated heterochromatin domain in mouse embryo fibroblasts (MEF)s. To do this, we have constructed a side-by-side comparison of wild-type (CpGFull) and CpG-depleted (CpGDep) promoter-driven reporter constructs in the context of the Chromatin in vivo Assay (CiA), which uses chemically-induced proximity (CIP) to tether the chromoshadow domain of HP1α (csHP1α) to a fluorescent reporter gene in a reversible, chemically-dependent manner. By comparing the response of CpGFull and CpGDep reporter constructs, we discovered that the heterochromatin formation by recruitment of csHP1α is unaffected by the underlying CpG dinucleotide content of the promoter, as measured by the velocity of gene silencing or enrichment of H3K9me3 at the silenced gene. However, recovery from long-term silencing is measurably faster in the CpG-depleted reporter lines. These data provide evidence that the stability of the HP1 heterochromatin domain is reliant on the underlying DNA sequence. Moreover, these cell lines represent a new modular system with which to study the effect of the underlying DNA sequences on the efficacy of epigenetic modifiers.
Highlights
The mammalian genomic landscape can be broadly divided into two regions defined by chromatin accessibility and a number of epigenetic marks
In order to determine the role of CpG dinucleotides in the kinetics of gene silencing and heterochromatin maintenance, we designed two reporter gene constructs with promoters of different CpG content
We used CRISPR/ Cas9 gene editing to insert a nucEGFP gene driven by the CpGFull or CpGDep promoter outside of the Hbb-γ gene in the β-globin locus in the mouse genome of large-T transformed mouse embryo fibroblasts (MEF) [43]
Summary
The mammalian genomic landscape can be broadly divided into two regions defined by chromatin accessibility and a number of epigenetic marks. Multiple distinct epigenetic pathways are in place to ensure that heterochromatinized regions of the genome remain transcriptionally silenced after successive rounds of cell division. Heterochromatin is observed transiently at the sites of double-stranded breaks and is important for the Ataxia Telangiectasia Mutated (ATM) double-stranded break repair pathway [9,10]. These markers of heterochromatin, like many other epigenetic modifications, have been found perturbed in human disease [11,12,13,14]. Heterochromatin patterning is very closely connected to another well-studied mark of heterochromatin that is intrinsically tied to the underlying DNA sequence, DNA methylation
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