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Abstract
BackgroundProtein posttranslational modifications (PTMs) occur broadly in the human proteome, and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylation sites among histone and nonhistone proteins have been characterized; however, the list of readers associated with them is incomplete.ResultsThis study introduces a modified yeast three-hybrid system (Y3H) to screen for methyllysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like the chromodomains of CBX1 or MPP8 as prey. We next conducted an unbiased screen using a library composed of human-specific open reading frames. It led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei.ConclusionsOur approach introduces a cost-effective method for screening reading domains binding to histone and nonhistone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the power of the Y3H assay which will allow for proteome-wide screens of PTM readers.
Highlights
Protein posttranslational modifications (PTMs) occur broadly in the human proteome, and their bio‐ logical outcome is often mediated indirectly by reader proteins that bind to modified proteins and trigger downstream effects
We demonstrate the proof of principle of the method, and, more importantly, we show that an unbiased screen using a library composed of human-specific open reading frames led to the identification of already known lysine methylation-dependent readers and novel methyllysine reader candidates
We have used a short histone H3N-terminal tail construct as bait, as we aimed to screen for H3K9me3 methylation-specific readers
Summary
Protein posttranslational modifications (PTMs) occur broadly in the human proteome, and their bio‐ logical outcome is often mediated indirectly by reader proteins that bind to modified proteins and trigger downstream effects. Different lysine residues can be subjected to modification by one, two, or three methyl groups at their ε-amine group, and each methylation state potentially has distinct functions in regulating transcription and chromatin compaction [3, 5]. Methylation does not substantially alter the biophysical properties of the lysine side chain, but it serves as a docking site for binding proteins called ‘readers’ that exert their function on chromatin [4, 6]. Depending on the methylation level and location of the modified residue, ‘readers’ trigger various effects including, but not limited to, regulation of chromatin compaction, recruiting other chromatin complexes or factors involved in DNA
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