Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Other
Summary
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. Further, our data support that CTCF mediates transcriptional insulator function through enhancer-blocking but not direct facultative heterochromatin barrier activity. Beyond defining the functions of CTCF in chromosome folding these results provide new fundamental insights into the rules governing mammalian genome organization.
Overall design
mouse ES cells were engineered to harbor an auxin-inducible degron (AID) tag at both endogenous alleles of CTCF. A transgene encoding the auxin-binding F-box protein Tir1 was subsequently introduce, so that adding auxin to the culture media leads to rapid (hours) and reversible degradation of CTCF. Consequences of acute loss of CTCF and its restoration were investigated using ChIP-seq, RNA-seq and high-throughput Chromosome Conformation Capture (5C and Hi-C).
Less...
More...