Study of artificial chromosomes by biologists sheds light on gene therapies

A research team led by Dr. Karen Wing Yee Yuen, associate professor at the School of Biological Sciences at the University of Hong Kong (HKU), has revealed the mechanism of artificial chromosome (AC) formation in embryos of the model organism Caenorhabditis elegans, a transparent nematode 1 mm long.

The results were published as two consecutive articles in the scientific journal Nucleic Acid Research. The studies provided information on the mechanisms of DNA assembly, the formation of new centromeres, and facilitated the engineering of CAs for cloning and gene therapy. In summary, Dr Karen Yuen and Dr Zhongyang Lin, postdoctoral researcher, discovered cellular proteins in vivo (inside a living organism) used to process foreign naked DNA fragments to form an artificial chromosome containing chromatin and dissected the molecular mechanisms of how foreign DNA of size kb can be assembled into an artificial chromosome larger than 10 megabases in C. elegans.

What are artificial chromosomes and why do they hold the key to the medicine of the future?

Essentially, our DNA is meticulously packaged by proteins to make up chromatin. If DNA were like a thread, these proteins are the coils around which the DNA thread winds to stay organized and tidy inside a microscopic cell. However, what will happen when a foreign, naked strand of DNA without a spool is introduced into the environment? Interestingly, the cell is equipped to provide this new thread with its own self-made spools, allowing this bare DNA thread to be stably maintained in the cellular environment as part of the new repertoire of the cell. We call this process the formation of artificial chromosomes (AC).

Among the useful applications of artificial chromosomes, one of the most interesting prospects is gene therapy. For example, chronic and fatal cystic fibrosis (CF) of the lungs is caused by a mutation in the CFTR gene and is currently an incurable disease. Scientists have studied the use of bacterial and yeast artificial chromosomes (BAC and YAC) as a vector or transporter to express the normal and functional CFTR gene and overcome defective CFTR expression in patients’ cells.

As in life, to manipulate something, you first have to understand it. In order to design artificial chromosomes, we must first learn how they are formed and maintained.

How new chromosomes are formed and maintained, and the importance of the centromere

Almost two trillion cells divide every day in the average human body. This means that two trillion cells have to make a perfect copy of themselves each time. The cost of cell division which is not beyond reproach is without a doubt the worst enemy of humanity to date: cancer, in which many are characterized by chromosomal instability. An important player in ensuring the faithful inheritance of our chromosomes during cell division is the centromere.

The centromere is a specialized region on each chromosome that connects the chromosome with the microtubules of the spindle to orchestrate the segregation of chromosomes in each cell division. In some cancer cells, centromeres can become inactivated or lost due to chromosome rearrangements and bypass chromosome loss by forming a new centromere on a random ectopic region. So far, not much is known about the formation of new centromeres (neocentromers), although it is implicated in chromosomal instability and a driver of tumorigenesis. This is because the formation of neocentromers is notoriously difficult to study, as the process of establishing neocentromers has been difficult to observe, as they are only determined when developmental disorders or cancer occur and genomic analyzes are performed. In other words, new centromeres are often detected long after their formation and stabilization.

To conduct studies on the formation of centromeres, Dr. Yuen’s team used a simple and straightforward method: microinjection, which was developed almost 30 years ago. In other species, such as humans, foreign DNA is recognized and mostly excluded and therefore not propagated into future generations as a self-defense mechanism. Surprisingly, C. elegans is one of the few species that allows foreign DNA, completely devoid of any C. elegans DNA sequence, to fuse into a large, artificial chromosome the size of a megabase. Simply put, while C. elegans can construct an artificial chromosome without a requirement for a native sequence, the same cannot be said of other artificial chromosomes from other species such as humans (HACs), which require certain sequences of Human DNA to build and propagate as an artificial chromosome.

To further study this unique characteristic, the team developed an in vivo fluorescent system to visualize artificial chromosomes in real time. Dr. Yuen’s lab is using this artificial chromosome segregation test as a functional indicator of de novo (from the start) centromere formation to study the factors that affect the establishment of de novo centromeres. Dr Lin identified the histone chaperone RbAp46 / 48^LIN-53 and HAT-1 acetyltransferase as essential for the formation of centromeres. Here, C. elegans acts as a robust model due to its transparent embryos which facilitate imaging, but also its rare nature to effectively induce the formation of de novo centromeres and faithfully separate new artificial chromosomes within a few cell cycles in them. embryos.

Current studies of artificial chromosomes provide new information on the chromosomal processes necessary for de novo formation of centromeres and maintenance of chromosomes. Dr. Yuen’s team revealed the in vivo biological processes necessary for exogenous DNA to become a stable propagating artificial chromosome and disentangle the hierarchy in assembling a de novo centromere from foreign DNA fragments. To further dissect the unique characteristic of C. elegans in the adoption of foreign DNA sequences, Dr. Yuen’s team also explored whether this phenomenon is bound by rules, that is, by manipulating the composition. , the complexity and length of the DNA sequence to observe the preferences for the construction of an artificial chromosome in C. elegans.

Thanks to these approaches, we are now able to observe and systematically compare how a new centromere is established on an artificial chromosome and how a pre-existing centromere is maintained on the endogenous chromosomes of C. elegans.

Can worms’ artificial chromosomes be the answer to gene therapy?

Finally, how are worms relevant to humans? While the order in which genes are arranged on a chromosome may be consistent between different closely related species, repositioning of the centromere also occurs throughout evolution. Therefore, the process of formation of new centromeres can act as a motor of disease but also serves as a marker of evolution. In addition, the results of these studies could help advance the field of synthetic biology by exploring how certain characteristics can be designed to optimize the establishment of an artificial chromosome by improving the efficiency of de novo centromere formation through to precise segregation to improve the applications of ACs with such high capacity, faithful vectors for cloning and gene therapy.