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.