Chromatin insulators demarcate expression domains by blocking the cis effects of 
enhancers or silencers in a position-dependent manner. We show that the 
chromatin insulator protein CTCF carries a post-translational modification: 
poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a 
poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal 
allele of the insulator domain in the H19 imprinting control region, requires 
the bases that are essential for interaction with CTCF. Chromatin 
immunoprecipitation-on-chip analysis documented that the link between CTCF and 
poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An 
insulator trap assay showed that the insulator function of most of these CTCF 
target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) 
polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin 
insulator properties to CTCF at both imprinted and nonimprinted loci, which has 
implications for the regulation of expression domains and their demise in 
pathological lesions.