Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, our data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization. [Display omitted] •Acute depletion of endogenous CTCF in ESCs with an auxin-inducible degron•CTCF instructs chromatin loops and TAD insulation genome wide•Disrupting TAD insulation does not impact higher-order genomic compartmentalization•CTCF depletion impacts transcriptional activity but not spread of H3K27me3 domains Dissecting out the roles of CTCF on genome looping, organization into TADs, and overall chromatin compartmentalization reveals fine layers of regulation.