A piece-by-piece replica of chromatin – PhD defense Bruna Eckhardt

Before a human cell can divide, it must copy its entire genome: about 2 meters of DNA, a massive library of biological instructions. To fit inside the nucleus, the cell’s control center, DNA is tightly packed into a structure called chromatin. This is done by wrapping DNA around proteins called histones, much like thread wrapped around a spool. These ‘spools’ are called nucleosomes. This dense packaging is essential for normal cell function, but it also creates a challenge. To copy the DNA, the cell’s replication machinery must temporarily ‘unwrap’ it, disrupting the chromatin structure. This disruption can threaten cell identity –the molecular memory that tells a cell whether it should be a skin cell, a heart cell, or a neuron.

How this memory is preserved when cells divide is a central question in epigenetics. Errors in this process can lead to genome instability, disrupted gene regulation, and diseases such as cancer. For her thesis, Bruna investigated how chromatin organization is safely disrupted and then restored during DNA replication.

Cell-in-a-tube

To learn how cells copy both their DNA and its packaging, Bruna rebuilt DNA replication from scratch in a test tube. This allowed her and her colleagues to study the molecular ‘gears’ involved in a way that is practically impossible inside a living organism. Building on systems developed by other groups, Bruna advanced the approach by adding chromatin assembly processes that had not previously been recreated in this way.

This was no easy feat, since the system depended on many purified proteins. This required careful planning and protein stock management, for which Bruna received tremendous help from technician Inge. Working with so many components was a challenge: small changes could strongly affect the outcome of an experiment. “Troubleshooting these complex systems was often frustrating, and both mentally and physically demanding,” Bruna reflects.

A tale of two strands

Using the cell-in-a-tube system, Bruna and her colleagues were the first to look closely at the chromatin scaffolding on the two newly copied DNA strands: the leading and the lagging strand, which are copied in different ways. The leading strand is copied continuously, while the lagging strand is copied in shorter pieces that are later joined together. The team discovered that chromatin organization also differs between the two newly copied strands. The leading strand was organized and neat, with evenly spaced nucleosomes. The lagging strand, by contrast, was more chaotic, with irregular spacing and incomplete structures. This suggests that cells need additional processes to restore a uniform chromatin structure before division. It also shows that DNA replication and chromatin assembly are more closely connected than previously thought.

Bruna collaborated with the laboratory of Vijay Ramani at the Gladstone Institutes and University of California, San Francisco. Coordinating a collaboration on the other side of the world brought its own challenges. Surprisingly, the biggest challenge was shipping samples reliably: one package containing six months of work by Bruna and Inge was lost for two weeks and could no longer be used when recovered.

The traffic controller activity of CAF-1

CAF-1 is an important protein complex for chromatin assembly. It shuttles histones and helps reassemble nucleosomes on the newly copied DNA strands. Bruna and her colleagues found that while CAF-1 builds chromatin on both strands, its interplay with the DNA-copying machinery differs between them. On the leading strand, DNA replication and chromatin assembly do not happen at the same time. On the lagging strand, the two processes are more closely coupled. They also found that chromatin assembly by CAF-1 can influence how fast DNA is copied. In other words, rebuilding chromatin can work like a ‘speed governor’ for DNA replication.

By developing new ways to study how chromatin is assembled and inherited during cell division, Bruna’s work provides an unprecedented level of detail into a process that is essential for healthy cell function. These insights may help other researchers investigate how errors in chromatin organization arise, how they affect gene regulation and genome stability, and why these processes are often disrupted in diseases such as cancer.

Bruna looks back fondly on her PhD journey: “I believe that a PhD serves not only the purpose of giving you a title, or knowledge, or even forming a great scientist: it shapes character.” She is grateful for all the support she received from colleagues, friends and family, and especially from her supervisor Francesca Mattiroli. Her advice for future PhD students? “Find the driving force that will carry you to the finish line, but learn to not define yourself entirely by that pursuit.”