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Authors: Rawłuszko-Wieczorek, Agnieszka Anna
Knodel, Franziska
Tamas, Raluca
Dhayalan, Arunkumar
Jeltsch, Albert
Title: Identification of protein lysine methylation readers with a yeast three-hybrid approach
Issue Date: 2018 Zeitschriftenartikel 12 Epigenetics & chromatin 11 (2018), No. 4
ISSN: 1756-8935
Abstract: Background: Protein 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 methylations sites among histone and non-histone proteins have been characterized, however, the list of readers associated with them is incomplete. Results: This study introduces a modified yeast-three-hybrid system (Y3H) to screen for methyl-lysine 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 chromodomain of CBX1 or MPP8 as prey. 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 of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei. Conclusions: Our approach introduces a cost-effective method for screening reading domains binding to histone and non-histone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the high capability of the Y3H assay which will allow for proteome wide screens of PTM readers.
Appears in Collections:03 Fakultät Chemie

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