Proteome-wide identification and molecular characterization of PHD-finger proteins recognizing H3K4me3 in arabidopsis

Abstract

In yeast and animals, tri- and di-methylation of histone H3 at lysine 4 (H3K4me3/2) are markers of transcriptionally active genes that have recently been shown to be the primary ligands for the plant homeodomain (PHD) finger. However, PHD fingers able to bind to H3K4me3/2 have not been identified in plants. Here, I identify 83 canonical PHD fingers in the Arabidopsis proteome database that are supported by both SMART and Pfam prediction. Among these, In vitro binding experiments showed that PHD fingers of Arabidopsis ING homologue (AtING) and alfin1-like (AL) proteins were able to bind to H3K4me3/2, of which predicted tertiary structures are highly similar to those in human ING2 and BPTF. ING proteins are found in all eukaryotes including yeast, animals, and plants, whereas AL proteins exist only in plants. In addition, mutational analysis confirmed that a predicted aromatic cage and a specific conserved acidic residue are both crucial for binding to H3K4me3/2. I also demonstrated that AtING and AL proteins are nuclear proteins expressed in various tissues of the Arabidopsis plant. Finally, physiological studies using T-DNA knock-out plants showed that AL proteins might play role in salt stress signaling, which is reported in alfalfa alfin1 studies. Thus, I propose that ING and AL proteins are nuclear proteins that are involved in chromatin regulation by binding to H3K4me3/2, the active histone markers, in plants.