Chromatin insulators separate active transcriptional domains and block the 
spread of heterochromatin in the genome. Studies on the chicken hypersensitive 
site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 
motifs in the proximal 250 bp of cHS4, termed the "core", which provide enhancer 
blocking activity and reduce position effects. However, the core alone does not 
insulate viral vectors effectively. The full-length cHS4 has excellent 
insulating properties, but its large size severely compromises vector titers. We 
performed a structure-function analysis of cHS4 flanking lentivirus-vectors and 
analyzed transgene expression in the clonal progeny of hematopoietic stem cells 
and epigenetic changes in cHS4 and the transgene promoter. We found that the 
core only reduced the clonal variegation in expression. Unique insulator 
activity resided in the distal 400 bp cHS4 sequences, which when combined with 
the core, restored full insulator activity and open chromatin marks over the 
transgene promoter and the insulator. These data consolidate the known 
insulating activity of the canonical 5' core with a novel 3' 400 bp element with 
properties similar to the core. Together, they have excellent insulating 
properties and viral titers. Our data have important implications in 
understanding the molecular basis of insulator function and design of gene 
therapy vectors.