Proteins containing defined recognition modules mediate readout and translation 
of histone modifications. These factors are thought to initiate downstream 
signaling events regulating chromatin structure and function. We identified 
CDYL1 as an interaction partner of histone H3 trimethylated on lysine 9 
(H3K9me3). CDYL1 belongs to a family of chromodomain factors found in 
vertebrates. We show that three different splicing variants of CDYL1, a, b, and 
c, are differentially expressed in various tissues with CDYL1b being the most 
abundant variant. Although all three splicing variants share a common C-terminal 
enoyl-CoA hydratase-like domain, only CDYL1b contains a functional chromodomain 
implicated in H3K9me3 binding. A splicing event introducing an N-terminal 
extension right at the beginning of the chromodomain of CDYL1a inactivates its 
chromodomain. CDYL1c does not contain a chromodomain at all. Although CDYL1b 
displays binding affinity to methyl-lysine residues in different sequence 
context similar to chromodomains in other chromatin factors, we demonstrate that 
the CDYL1b chromodomain/H3K9me3 interaction is necessary but not sufficient for 
association of the factor with heterochromatin. Indeed, multimerization of the 
protein via the enoyl-CoA hydratase-like domain is essential for H3K9me3 
chromatin binding in vitro and heterochromatin localization in vivo. In 
agreement, overexpression of CDYL1c that can multimerize, but does not interact 
with H3K9me3 can displace CDYL1b from heterochromatin. Our results imply that 
multimeric binding to H3K9me3 by CDYL1b homomeric complexes is essential for 
efficient chromatin targeting. We suggest that similar multivalent binding 
stably anchors other histone modification binding factors on their target 
chromatin regions.