Evolved Readers of 5-Carboxylcytosine CpG Dyads Reveal a High Versatility of the Methyl-CpG-Binding Domain for Recognition of Noncanonical Epigenetic Marks.

Mammalian genomes are regulated by epigenetic cytosine (C) modifications in palindromic CpG dyads. Including canonical cytosine 5-methylation (mC), a total of four different 5-modifications can theoretically co-exist in the two strands of a CpG, giving rise to a complex array of combinatorial marks with unique regulatory potentials. While tailored readers for individual marks could serve as versatile tools to study their functions, it has been unclear whether a natural protein scaffold would allow selective recognition of marks that vastly differ from canonical, symmetrically methylated CpGs. We conduct directed evolution experiments to generate readers of 5-carboxylcytosine (caC) dyads based on the methyl-CpG-binding domain (MBD), the widely conserved natural reader of mC. Despite the stark steric and chemical differences to mC, we discover highly selective, low nanomolar binders of symmetric and asymmetric caC-dyads. Together with mutational and modelling studies, our findings reveal a striking evolutionary flexibility of the MBD scaffold, allowing it to completely abandon its conserved mC recognition mode in favour of noncanonical dyad recognition, highlighting its potential for epigenetic reader design.

Light-Activatable MBD-Readers of 5-Methylcytosine Reveal Domain-Dependent Chromatin Association Kinetics In Vivo.

5-Methylcytosine (5mC) is the central epigenetic mark of mammalian DNA, and plays fundamental roles in chromatin regulation. 5mC is dynamically read and translated into regulatory outputs by methyl-CpG-binding domain (MBD) proteins. These multidomain readers recognize 5mC via an MBD domain, and undergo additional domain-dependent interactions with multiple additional chromatin components. However, studying this dynamic process is limited by a lack of methods to conditionally control the 5mC affinity of MBD readers in cells. Light-control of MBD association to chromatin by genetically encoding a photocaged serine at the MBD-DNA interface is reported. The authors study the association of MBD1 to mouse pericentromeres, dependent on its CxxC3 and transcriptional repressor domains (TRD) which interact with unmethylated CpG and heterochromatin-associated complexes, respectively. Both domains significantly modulate association kinetics, arguing for a model in which the CxxC3 delays methylation responses of MBD1 by holding it at unmethylated loci, whereas the TRD promotes responses by aiding heterochromatin association is studied. Their approach offers otherwise inaccessible kinetic insights into the domain-specific regulation of a central MBD reader, and sets the basis for further unravelling how the integration of MBDs into complex heterochromatin interaction networks control the kinetics of 5mC reading and translation into altered chromatin states.

Correction: A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

[This corrects the article DOI: 10.1039/D2MD00186A.].