Abstract
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.
Post-translational modifications (PTM) of histones, the proteins around which DNA is wrapped in chromatin, have been implicated in different biological processes ranging from transcriptional regulation to cell cycle progression. Many histone PTMs recruit specific proteins that translate their function into biological outcomes. Understanding the binding mode and molecular biology of these factors is key for our comprehension of epigenetic processes. In this study, we found that the
Caenorhabditis elegans
LIN-61 protein specifically interacts with particular PTMs on histone H3, di- and trimethylation of lysine 9 (H3K9me2/3), which are implicated in transcriptional repression. LIN-61 contains so-called malignant brain tumor repeats (MBT), which have been found to bind histone methyl-lysine residues in other proteins and model systems. However, these interactions are limited to the mono- and dimethylated states, and there is little to no sequence specificity for particular histone lysine residues. Importantly, H3K9me2/3 binding by LIN-61 is essential for the function of this factor within the synMuv pathway of
C. elegans
vulva cell fate determination. Besides identifying the first sequence and methylation state specific MBT protein, our studies define novel functions of LIN-61 and manifest a role of H3K9me2/3 in the synMuv pathway.