Striking clocks, such as cuckoo clocks and automaton clocks, are not the only mechanical machines that set animated figures into motion at regular intervals. Chromatin, consisting of DNA and its associated material, also serves as a timer, changing the orientations of its internal parts to trigger actions such as replication.

Until recently, the nature and locations of these internal parts has been as obscure as the springs and gears behind a clockface. But now, thanks to the work of scientists based at Florida State University (FSU), the chromosome’s replication clockwork has been exposed.

A scientific team led by David Gilbert, Ph.D., a professor of molecular biology at FSU, revealed that there are specific points along the DNA molecule that control replication. To do so, the team systematically deleted genomic elements to identify three early replication control elements (ERCEs). When the ERCEs are removed, the large-scale 3D structure of the DNA molecule changes, and the timing of DNA replication is shifted.

Detailed findings appeared December 28 in the journal Cell, in an article titled, “Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication.” The article contains information that may guide future studies probing the significance of replication timing aberrations in human disease. It may also contribute to innovations in chromosome domain engineering that will advance chromosome biology.

In particular, the article describes how CRIPR/Cas9-mediated chromosome engineering was used to generate deletions and inversions within and between adjacent domains. These genomic interventions were designed to perturb replication timing, topologically associating domain (TAD) structure, and subnuclear compartmentalization.

“Deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late replication timing shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription,” the article’s authors wrote. “The dispensability of TAD boundaries and the necessity of these ‘early replication control elements’ (ERCEs) was validated by deletions and inversions at additional domains.”

The FSU team ran close to a hundred genetic mutations on DNA molecules, hoping to see some sort of result that would better explain how the replication process worked. At a point of frustration, Dr. Gilbert said that the FSU team came up with a “hail Mary” attempt.

The researchers examined a single segment of the DNA in the highest possible 3D resolution and saw three sequences along the DNA molecule touching each other frequently. The researchers then used CRISPR, a sophisticated gene editing technology, to remove these three areas simultaneously.

And with that, they found that these three elements together were the key to DNA replication.

Graphical abstract showing how genomic features called early replication control elements (ERCEs) orchestrate domain-wide replication timing, A/B compartmentalization, topologically associating domain (TAD) architecture, and transcription. [Cell]

“Removing these elements shifted the segment’s replication time from the very beginning to the very end of the process,” Dr. Gilbert said. “This was one of those moments where just one result knocks your socks off.”

In addition to the effect on replication timing, the removal of the three elements caused the 3D structure of the DNA molecule to change dramatically.

“We have for the first time pinpointed specific DNA sequences in the genome that regulate chromatin structure and replication timing,” noted doctoral student Jiao Sima, the lead author of the current study. “These results reflect one possible model of how DNA folds inside cells and how these folding patterns could impact the hereditary materials’ function.”

Greater understanding of how DNA replication is regulated opens new paths of research in genetics. When replication timing is altered, it can completely change how the genetic information of a cell is interpreted.

This could become crucial information as scientists tackle complicated diseases where the replication timing is disrupted.

“If you duplicate at a different place and time, you might assemble a completely different structure,” Dr. Gilbert explained. “A cell has different things available to it at different times. Changing when something replicates changes the packaging of the genetic information.”

“Our results,” the authors of the Cell article concluded, “demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.

“It’s been quite a mystery,” Dr. Gilbert commented. “Replication seemed resilient to everything we tried to do to perturb it. We’ve described it in detail, shown it changes in different cell types, and that it is disrupted in disease. But until now, we couldn’t find that final piece, the control elements or the DNA sequences that control it.”

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