NEW YORK (GenomeWeb) – Researchers led by a team at the Gladstone Institutes in San Francisco have used the auxin-inducible degron system to demonstrate that the protein CTCF plays a critical role in determining how genes are organized during the chromosome folding process.
Researchers had previously established that chromosomes are coiled into loops and then further organized into many large domains called topologically associating domains, or TADs. "Imagine TADs are like adjoining rooms: like the genes in each TAD, people in each room can talk to one another, but not to people in the next room," Elphège Nora, first author on the study published today in Cell, said in a statement.
The Gladstone team had already established that TADs package genes together and insulate them from one another, but didn't know how TAD organization was controlled. "[CTCF] is a fascinating protein," added senior author Benoit Bruneau. "It can be found at the boundaries of TAD domains, and was previously thought to be involved in many aspects of chromosome organization. We wanted to see what would happen to the structure of chromosomes if we removed all the CTCF from cells."
Bruneau, Nora, and their colleagues decided to use the auxin-inducible degron system to deplete CTCF proteins in embryonic mouse stem cells to an undetectable level. The researchers then measured changes in chromosome organization by performing high-throughput chromosome conformation capture in untreated cells, cells treated for two days with auxin, and cells that were treated then washed after two days. They also confirmed their findings were consistent throughout the genome, and not just at a single chromosome location, by performing Hi-C in untreated, two-day treated, and washed off two-day treated cells.
"We looked at a level of organization called compartmentalization, which separates active and inactive genes within a cell nucleus," said Nora. "We used to think that boundaries of TAD domains were a prerequisite for the organization of these compartments."
But this was not the case, according to Bruneau. "When we deleted the CTCF protein, which caused TAD boundaries to disappear, we saw no effect on the organization of the larger compartments," he said. "This interesting finding revealed that CTCF and TAD structure are not required for compartmentalization but, rather, that an independent mechanism is responsible for this chromosome organization."
Bruneau and his colleagues also noted that their data supported the conclusion that CTCF mediates transcriptional insulator function thorough enhancer blocking, but not as a direct barrier to heterochromatin spreading.
The team believes that the next steps will be to investigate the molecular details of these processes and how they modulate transcriptional patterning, as well as other nuclear processes.