MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3 binding proteins, LIN-61 and HPL-2, as well as the H3K9MT MET-2 in distinct developmental pathways.
Bibliographical noteFunding Information:
Monika Jedrusik-Bode made the initial observation of LIN-61–H3K9me2/3 interaction and provided valuable comments on the progress of this work as well as on the content of the manuscript. We thank the Proteomics group at the Max Planck Institute for Biophysical Chemistry of Henning Urlaub for excellent mass spectrometry analysis. We are grateful to Francesca Palladino and Robert Horvitz for C. elegans strains and reagents. The National BioResource Project made the lin-61(tm2649) C. elegans strain. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. We thank Christoph Biesemann and Alexandra Stützer for help with cloning expression constructs and Martina Wirth and Henriette Franz for establishing C. elegans and peptide affinity purification procedures. We further acknowledge Szabolcs Sörös and Adrian Schomburg for help with protein purification, as well as Kerstin Mosch for valuable comments on the progress of this study.
ASJC Scopus subject areas
- Ecology, Evolution, Behavior and Systematics
- Molecular Biology
- Cancer Research