The contribution of human subtelomeric DNA and chromatin organization to 
telomere integrity and chromosome end protection is not yet understood in 
molecular detail. Here, we show by ChIP-Seq that most human subtelomeres contain 
a CTCF- and cohesin-binding site within ∼1-2 kb of the TTAGGG repeat tract and 
adjacent to a CpG-islands implicated in TERRA transcription control. ChIP-Seq 
also revealed that RNA polymerase II (RNAPII) was enriched at sites adjacent to 
the CTCF sites and extending towards the telomere repeat tracts. Mutation of 
CTCF-binding sites in plasmid-borne promoters reduced transcriptional activity 
in an orientation-dependent manner. Depletion of CTCF by shRNA led to a decrease 
in TERRA transcription, and a loss of cohesin and RNAPII binding to the 
subtelomeres. Depletion of either CTCF or cohesin subunit Rad21 caused 
telomere-induced DNA damage foci (TIF) formation, and destabilized TRF1 and TRF2 
binding to the TTAGGG proximal subtelomere DNA. These findings indicate that 
CTCF and cohesin are integral components of most human subtelomeres, and 
important for the regulation of TERRA transcription and telomere end protection.