Direct analysis of transcription factor protected cfDNA in plasma by ChIP-seq: Measurement of altered CTCF binding in cancer is a novel biomarker for liquid biopsy

For the first time, we have isolated endogenous plasma cell free CTCF-DNA (cfCTCF-DNA) nucleoproteins by chromatin immunoprecipitation (ChIP) and sequenced the associated cfDNA (ChIP-Seq). The mean observed enrichment of plasma cfCTCF-DNA by ChIP was 180-fold with a mean CTCF recovery of 48%. Background cfDNA, primarily in the form of nucleosomes, was almost completely removed (> 99.7%). ChIP-seq analysis of cell line and tissue material is a powerful tool that has underpinned our understanding of epigenetic control and has elucidated tens of thousands of CTCF binding sites in the human genome. CTCF binding is altered in cancer and other diseases indicating the potential for use as a tissue based biomarker. In addition, the presence of cell free cfCTCF-DNA in plasma has been inferred in aggregate from cfDNA-footprinting and nucleosome positioning studies. However, these methods have provided little or no information on the occupancy of individual CTCF binding sites limiting their potential for biomarker use in liquid biopsy. We describe direct liquid biopsy analysis of CTCF occupancy of individual CTCF binding sites represented in plasma as short CTCF associated 35–80 base pair cfDNA fragments. We report the observation of several hundred cfCTCF-DNA binding site loci representing cancer-associated gain of occupancy CTCF binding site sequences present in the plasma of cancer patients but not present in the plasma of healthy subjects. Longer 135–180 base pair cfDNA fragments bearing the same sequences are found in healthy plasma samples, but these are nucleosome bound and are removed by CTCF ChIP. We conclude that plasma cfCTCF-DNA nucleoproteins represent a new class of liquid biopsy biomarkers that can be directly analyzed in plasma by ChIP-Seq. In addition, the method described may have wider application for analysis of the binding of some of the 370 cancer cell derived transcription factors, each with more than 10,000 binding sites in the human genome, whose activity has been detected in plasma by cfDNA nucleosome positioning analysis.