Scientafic!

My work on the mitotic inheritance of extrachromosomal DNA (ecDNA) in cancer is now available as a preprint on bioRxiv¹ (the study has now been published in the Journal of Biological Chemistry). In this post, I try to summarize the scientific process and a few of the main points of the study. I divided the post into a few sections: the question, the hypothesis, the foray into prometaphase spreads, the results, the conceptual framework, and the take home message.

The question

In this study, I asked the question of how ecDNA, which are common in cancer and contribute quite a bit to cancer pathogenesis, are inherited by daughter cells during mitosis, or cell division. In normal cells, essentially all of the genes are found on chromosomes, which are large molecules made up of DNA and protein (i.e. chromatin) that represent units of genetic inheritance. Importantly, chromosomes contain specialized regions called centromeres. In mitosis, mitotic spindles attach to these centromeres, allowing them to push and pull chromosomes to make sure they are properly distributed into the two newly formed daughter cells. The actions of spindles ensure that genetic material is 1) equally distributed into daughter cells and 2) inherited inside the nuclei of daughter cells, where genetic material normally resides.

In cancer cells, some cancer-causing oncogenes and other DNA elements are found on ecDNA rather than on chromosomes. When this happens, the cancer becomes more difficult to treat for reasons that are yet to be fully elucidated. Just like chromosomes, ecDNA are molecules composed of DNA and protein, except ecDNA are small and, notably, lack centromeres. During mitosis, therefore, ecDNA presumably do not interact directly with mitotic spindles the same way chromosomes do. As a result, studies have shown that ecDNA are not equally distributed into daughter cells during mitosis. This creates cancer heterogeneity, which may help cancers evade treatment. Interestingly, despite lacking centromeres, ecDNA are mostly inherited inside the nuclei of daughter cells after mitosis. How do they do this without centromeres? Well, it was observed nearly 50 years ago that during mitosis, ecDNA hitchhike on chromosomes as they are pulled by spindles into daughter cell nuclei. There appears to be a force that tethers ecDNA to chromosomes during mitosis, helping to ensure that they make it inside daughter cell nuclei along with the chromosomes. The identity of this “force” is only now beginning to be elucidated.

During mitosis, ecDNA tether to chromosomes to hitchhike their way into daughter cell nuclei. Note: I drew chromosomes here as being separated and distinct from one another inside the cell to simplify the depiction, but as I argue below, this is likely not the case.

I wanted to figure out the identity of this “force”. The idea is that if we disrupt this force and untether ecDNA from chromosomes during mitosis, the ecDNA will end up in the cytoplasm of newly formed daughter cells after cell division rather than the nuclei, where they are more prone to degradation by harsh cytosolic conditions and lack the machinery to faithfully replicate.

The hypothesis

I started with a simple hypothesis: ecDNA-chromosome tethering is not an ecDNA-specific phenomenon. Rather, perhaps all chromatin (molecules of DNA and protein, i.e. chromosomes and ecDNA) tether to each other during mitosis, including chromosomes with each other, as chromatin should be quite sticky during mitosis. This is because during mitosis, chromatin compacts into denser forms, which allows them to move more easily in the cell when pushed and pulled by spindles, in a process called mitotic chromatin compaction. During compaction, nucleosomes, the basic unit of chromatin, interact with each other more tightly, due to stronger nucleosome-nucleosome attractive forces, such as electrostatic and hydrophobic interactions. I hypothesized that these attractive forces act not only within a piece of chromatin (such as a chromosome) to compact it, but also between difference pieces of chromatin (such as chromosomes and ecDNA) to tether them to each other.

During mitosis, uncompacted chromatin (left) become compacted (right), due to increased electrostatic and hydrophobic forces acting between nucleosomes, which bring them closer to each other.

A brief foray into prometaphase spreads

While I was working on this hypothesis, I became interested in prometaphase spread preparations of cells. The prometaphase spread technique is frequently used in clinical cytogenetics to generate karyotypes from cells undergoing mitosis, because it allows all the chromosomes and other genetic elements within cells to be nicely separated from each other and distinctly visualized for identification and closer examination. However, exactly how the prometaphase spread technique achieves this is not clearly described. Interestingly, in prometaphase spreads of ecDNA-containing cells, the ecDNA are also nicely separated, or untethered, from each other and chromosomes. Therefore, I wondered how the prometaphase spread technique is able to untether ecDNA from chromosomes, as this may provide a clue as to the identity of the force tethering ecDNA to chromosomes

After some careful experiments, I figured out that during prometaphase spread preparations, the step where cells are incubated in a hypotonic solution is important for generating nicely separated and distinct chromosomes and ecDNA. Without this step, all of the chromosomes and ecDNA clump to each other, preventing any visual identification of individual chromosomes. Traditionally, the hypotonic solution incubation step is thought to increase the net inflow of water into cells via osmosis, causing cells and their contents to swell, which increases the size of chromosomes and gives them more room inside the cell to spread apart. I thought that an alternative explanation is that the net inflow of water into cells decreases the intracellular ion concentration, thereby disrupting the electrostatic and hydrophobic interactions among nucleosomes. This should cause chromosomes and ecDNA to decompact (become bigger) and untether from each other (if my hypothesis that chromatin compaction causes ecDNA-chromosome tethering is correct).

To test this hypothesis, I made prometaphase spread preparations of cells but skipped the step where cells are incubated in hypotonic solution. Instead, I treated cells with a chemical drug called an histone deacetylase (HDAC) inhibitor, which is known to decompact chromatin. If my hypothesis is correct that hypotonic solution incubation separates ecDNA and chromosomes by decompacting chromatin, then treatment with an HDAC inhibitor should do the same. To my pleasant surprise, this worked beautifully. Prometaphase spread preparations of cells produced with HDAC inhibitor treatment rather than hypotonic solution incubation resulted in nicely separated and distinct ecDNA and chromosomes. In fact, the spreads produced were of high enough quality for karyotype assembly.

Experimental results

Armed with the information I learned from prometaphase spread preparations, I next tested whether chromatin decompaction (via hypotonic solution incubation and HDAC inhibition) can untether ecDNA from chromosomes in mitotic cells in their native state (i.e. not prometaphase spreads) for better morphological and physiological preservation. Not surprisingly, it did. Also as predicted, at the end of mitosis, some of the untethered ecDNA wound up in the cytoplasm of the newly formed daughter cells, rather than inside the nuclei.

The analogy I like to use to think about this finding is that ecDNA sticks to chromosomes (and chromosomes stick to each other) similarly to the way dust sticks to our hands. The surface of skin is electrically charged and opposite charges found on dust allows it to stick to skin via electrostatic attraction. Water, which is polar (contains partial charges), disrupts this electrostatic attraction by interacting with the charges on skin and dust, preventing the two from interacting with each other. ecDNA and chromosomes are also electrically charged and stick to each other via electrostatic attraction, which is strengthened by the presence of ions within the cell. When cells are incubated in hypotonic solutions, water rushes into the cells, interacting with the charges on ecDNA and chromosomes and lowering the ionic concentration inside the cell, thereby disrupting the electrostatic interactions tethering ecDNA to chromosomes (and chromosomes to each other). HDAC inhibition, on the other hand, disrupts electrostatic ecDNA-chromosome interactions by directly removing their charges.

Soap works along with water to help us clean our hands. Soap is amphiphilic, meaning one end of soap molecules interacts with dirt and dust, while the other interacts with water. Thereby, soap surrounds particles of dirt and dust, preventing them from sticking to skin while helping water rinse them away. I wondered, then, whether there might be a “soap” that acts similarly to prevent ecDNA-chromosome tethering.

In fact, there is. Ki67 is a natural protein produced by cells that acts as a biological surfactant (i.e. soap) coating the surface of chromosomes during mitosis, helping to prevent chromosomes from sticking to each other too tightly. Like all soap, Ki67 is amphiphilic, with one end of the molecule preferentially interacting with chromatin (i.e. the surfaces of chromosomes) and the other end preferentially interacting with water (i.e. cytosol). I thought that Ki67 may coat the surface of ecDNA as well and help to prevent ecDNA-chromosome tethering. I tested this idea by increasing the concentration of Ki67 within cells. Again, to my delight, this effectively untethered ecDNA from chromosomes, just like in cells incubated in hypotonic solution or treated with HDAC inhibitors. Therefore, water and soap can quite literally wash ecDNA off of chromosomes during mitosis.

A proposed conceptual framework

The framework I use to think about this conceptually is based on colloidal and surface chemistry. In this framework, the mitotic cell is a colloidal suspension of particles in a liquid solution at two levels: the level of nucleosomes, where particle-particle interactions mediate mitotic chromatin compaction, and the level of large chromatin molecules (ecDNA and chromosomes), where particle-particle interactions mediate tethering.

The take home message

In this study, I show that mitotic chromatin compaction tethers molecules of chromatin to each other during mitosis, ensuring that pieces of chromatin lacking centromeres do not end up in the cytoplasm after cell division. This means that centromeres and specialized molecular machinery are not necessarily required for extra bits of chromatin to be inherited by daughter cell nuclei during mitosis. Notably, Ki67, acting as a surfactant, or soap, opposes chromatin-chromatin tethering mediated by mitotic chromatin compaction forces. This finding makes me wonder if cells evolved to express Ki67 as a soap to wash off extrachromosomal pieces of chromatin, such as viral DNA, from chromosomes, thereby helping to prevent unwanted genetic material from being inherited during mitosis. In this way, Ki67 may be a cellular defense against viruses and other foreign DNA. Food for thought (and future studies).

Reference

1. Yang, Lu M. 2025. Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei. bioRxiv 2025.08.21.671584; doi: https://doi.org/10.1101/2025.08.21.671584.
   Now published in the Journal of Biological Chemistry https://www.jbc.org/article/S0021-9258(25)02933-3/fulltext ↩︎