| Popis: |
The three-dimensional structure of chromatin regulates gene expression by facilitating contacts between promoters and regulatory elements, demarcating regions of activity and repression and compartmentalising the genome into Topologically Associating Domains (TADs). Architectural protein CTCF is an established mediator of chromatin conformation, however attempts to deplete CTCF have had inconsistent consequences on chromatin and gene expression, which has prevented full comprehension of its role. The aim of this work is to clarify CTCF function and regulation. We began by performing a transient (144hrs) RNAi knockdown to deplete CTCF in LNCaP prostate cancer cells. CTCF ChIP-seq following knockdown revealed that 2949/25505 CTCF sites reproducibly remained bound following RNAi. CTCF RNAi knockdown was next performed in IMR-90 normal lung fibroblasts and revealed the same persistent subset of CTCF sites. To investigate the functionality of these sites we performed CRISPR-Cas9n experiments on candidate persistent sites in LNCaP cells. We chose a locus with a repressive loop containing eight genes, which had lost CTCF binding at 10/12 sites, but had not undergone any changes in gene expression. CRISPR-Cas9 of the 2 persistent sites caused activation of all genes within the loop and concordant alteration to the loop structure when measured by 3C. Hi-C demonstrated a general maintenance of TADs and that persistent sites were located at TAD boundaries. Bioinformatic profiling showed that these sites are constitutively bound in cell types of both normal and disease conditions and derived from different germ layers. We have identified a novel subset of CTCF sites that are persistent following CTCF RNAi and have shown evidence that maintenance of these sites can impede changes to chromatin conformation and gene expression. These results help clarify the current ambiguity in understanding the function and regulation of CTCF. |
