Scientists have discovered that cells become temporarily easier to reprogramme just after they copy their DNA. This finding helps to explain why mature cells are easier to reprogramme during bursts of replication – and could improve future regenerative medicine.
For decades, scientists have learned how to chemically coax stem cells into becoming other types of cells: neurons with branching axons, the distinctive red doughnuts of blood cells or stringy cardiac cells.
Since 2006, they have also accomplished the reverse: reverting mature cells back into stem cells with seemingly unlimited potential. But for reasons scientists have not fully understood, this kind of cellular fate change mainly occurs during bursts of cell replication.
“Division seems to be something cells need to do in order to change what kind of cell they are,” explains Kathleen Stewart-Morgan, a biologist at the University of Copenhagen, Denmark who studies epigenetic reprogramming.
Now, researchers from the University of Copenhagen and the Danish Cancer Institute believe they have pinned down why: just after DNA replication, newly packed chromatin briefly becomes unusually accessible – meaning that the molecular machinery that controls gene activity can more easily reach the DNA. This creates a short-lived window in which cellular identity appears to be easier to rewrite.
The machinery responsible for repacking DNA after copying appears to work quickly but imperfectly, temporarily leaving key regions of DNA easier to access.
By distinguishing newly replicated DNA from older DNA, the researchers were able to directly test whether replication itself helps drive cell fate change – and compare chromatin accessibility before and after replication within the same cells.
Their findings, published in Stem Cell Reports, could help researchers improve methods for turning mature cells such as skin and hair cells back into stem cells. The work may also shed light on how uncontrolled replication reshapes cellular identity in cancer.
How cells repack their DNA
Every cell in the human body, from the light-sensing rods in your retina to your tastebuds, is built from an identical assembly manual. “The DNA in those cells is the same. What is different is which genes are expressed in those cells,” says co-author Stewart-Morgan.
The difference comes down to the epigenome, a system of microscopic chemical tags that acts like bookmarks in the cell’s assembly manual, marking which instructions should be read and which should stay silent.
Some epigenetic tags can be inherited, but the epigenome also shifts in response to environmental conditions such as food scarcity. Nevertheless, scientists still do not fully understand when DNA becomes accessible enough for these changes to occur.
“DNA is not naked in the nucleus of the cell – it is packaged into chromatin, all the proteins associated with DNA,” Stewart-Morgan explains. “That packaging keeps the DNA safe and protects it from breaking.”
Within the chromatin, strands of DNA are tightly wound around proteins called histones, like thread around a spool. Co-author Anja Groth, a molecular biologist who studies the inheritance of the epigenome at the Danish Cancer Institute, has spent decades studying how chromatin is unpacked and repacked during replication.
“Basically, the replication machinery has to pry apart the two strands of the DNA double helix in order to build a new copy,” Groth says. “As that happens, the histones are temporarily displaced and then recycled onto the newly synthesised DNA.”
But after replication, the newly copied DNA must quickly be wrapped in fresh histones. The next step is a little slap-dash – new histones stream into the replication machinery and are rapidly wrapped around the new DNA.
“We know from earlier work, our own and from our collaborators, that this chromatin looks packed, but it is much more disordered,” Groth notes. “That disorder may make the DNA easier for regulatory proteins to access.”
Something old, something newly copied
Because this hastily packed chromatin is relatively disordered, the researchers suspected that cells might briefly become more vulnerable to identity change.
To test the idea, the researchers needed a way to distinguish newly replicated chromatin from older chromatin to measure how easily regulatory proteins could elbow their way into different stretches of DNA to switch genes on or off.
“The cells are growing in a dish. In addition to the four DNA bases – A, C, G and T – we add a chemical analogue that looks a lot like thymidine, the T in ACGT, but is not actually thymidine,” Stewart-Morgan explains.
As cells copied their DNA, the newly synthesised strands incorporated the fake thymidine, while the older DNA retained the natural version. That enabled the researchers to distinguish newly copied DNA from pre-existing DNA.
“Using this gave us a new way into the question and provided the real advance in this study,” Stewart-Morgan says.
Stem cells, scissors and a smoking gun
In a technique called repli-ATAC-seq, which Stewart-Morgan and Groth first presented in a 2019 article, an enzyme snips out whatever stretches of DNA are exposed at different stages of the cell cycle.
“In an unbiased way, it is just trying to cut whatever it can find,” Stewart-Morgan says. Researchers can then sequence these tiny snippets of DNA to see which parts of the genome were physically exposed and accessible inside the cell.
Surveying these little snipped-out sections revealed a consistent pattern: stretches of chromatin that became more accessible during replication were repeatedly linked to genes involved in lineage change.
“We know from a lot of correlative work that these regions are the starts of genes and enhancer regions – stretches of DNA that help control whether genes are active or silent,” Stewart-Morgan says.
Reprogramming catches cells mid-copy
But what about the other direction – cellular reprogramming? The researchers looked to Yamanaka factor reprogramming, a Nobel-prize winning protocol developed by a Kyoto University scientist in 2006. Yamanaka factor reprogramming can transform humble skin or hair cells into stem cells using specific transcription factors – proteins that switch off genes tied to the cell’s current identity and reactivate genes normally active only in embryos.
Yamanaka factor reprogramming is generally a two-week process, the researchers say. “Serendipitously, we captured a time point early on” that served as a smoking gun, Stewart-Morgan adds.
“After 12 hours, the unreplicated chromatin still resembled the starting cells,” Stewart-Morgan says. “But the replicated chromatin had already shifted toward a more stem-cell-like state.”
The difference appeared within the same reprogramming experiment, linking replication more closely to identity change.
When the researchers blocked replication, both the accessibility changes and the shift toward reprogramming disappeared.
“That was the real demonstration that the act of replication itself creates this opportunity for reprogramming,” Stewart-Morgan says. The accessibility measurements, lineage-linked regions and replication-blocking experiments all pointed in the same direction.
“Replication paves the way for the binding of these key transcription factors,” Groth adds. “If replication does not happen, you remove the effect.”
Rewriting cellular identity
Stewart-Morgan and Groth think that the findings could help researchers better control how cells change identity – a longstanding goal in regenerative medicine.
“There is also really interesting emerging work on partial reprogramming, in which cells are pushed partway back toward a younger state without fully losing their identity,” Groth explains. However, being cavalier with boosting cell division can have its own consequences, such as in the runaway cell growth in cancer, she cautions.
By the same token, these lessons could potentially be applied to diseases driven by runaway cell division, including cancer, they say.
“Once you understand which process is sort of the gatekeeper, you can start thinking, ‘is there something we can go in and manipulate to change that and maybe make higher accessibility after replication?’” Groth says. “It is not only about controlling whether cells divide, but potentially manipulating replication itself,” Groth concludes.
