1 research outputs found
Tandem Histone-Binding Domains Enhance the Activity of a Synthetic Chromatin Effector
Fusion proteins that
specifically interact with biochemical marks
on chromosomes represent a new class of synthetic transcriptional
regulators that decode cell state information rather than DNA sequences.
In multicellular organisms, information relevant to cell state, tissue
identity, and oncogenesis is often encoded as biochemical modifications
of histones, which are bound to DNA in eukaryotic nuclei and regulate
gene expression states. We have previously reported the development
and validation of the “polycomb-based transcription factor”
(PcTF), a fusion protein that recognizes histone modifications through
a protein–protein interaction between its polycomb chromodomain
(PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at
genomic sites. We demonstrated that PcTF activates genes at methyl-histone-enriched
loci in cancer-derived cell lines. However, PcTF induces modest activation
of a methyl-histone associated reporter compared to a DNA-binding
activator. Therefore, we modified PcTF to enhance its binding avidity.
Here, we demonstrate the activity of a modified regulator called Pc<sub>2</sub>TF, which has two tandem copies of the H3K27me3-binding PCD
at the N-terminus. Pc<sub>2</sub>TF has a smaller apparent dissociation
constant value <i>in vitro</i> and shows enhanced gene activation
in HEK293 cells compared to PcTF. These results provide compelling
evidence that the intrinsic histone-binding activity of the PCD motif
can be used to tune the activity of synthetic histone-binding transcriptional
regulators