Human artificial chromosomes (HACs) could be useful tools for both understanding how mammalian chromosomes function and creating synthetic biological systems, but for the last 20 years, they have been limited by an inefficient artificial centromere. In the journal Cell on July 25th, researchers announce that they have made progress on this key component.
"The centromere used to be called the black box of the chromosome," says Ben Black, professor of biochemistry and biophysics at the University of Pennsylvania. "If you're studying any kind of biological process, you want to be able to build it, and that's where we've made progress here."
In mammals, centromeres--the central point of the X-shaped chromosome--ensure that a chromosome is inherited when a cell divides, acting as an anchor for the spindle fibers that pull the duplicated chromosome in half. The genetic sequence of a natural human centromere is thousands of repetitions of a 171-base-pair sequence. Centromeric DNA must also be modified epigenetically in the cell to function properly. These epigenetic marks (protein and chemical tags along the DNA) are thought to be established at centromeres by the human CENP proteins.
First-generation HACs have relied on both the repetitive centromere sequence and CENP-B. But the repetitive sequence make centromeres tricky to clone for study in the lab. Therefore, "all of the synthetic chromosomes that have been recently reported use approaches that intentionally remove repetitive elements," Black says, making it so far impossible to transition the techniques that work in yeast artificial chromosomes to HACs.
Black's team has now created two new HACs: neither use CENP-B, and one is not repetitive. "We wanted to see if we can break the rules by bestowing the DNA we put into the cell with epigenetic markers from the get go," says Black. Their improvements remove the requirement for CENP-B, make the HACs more reliably inherited in cell culture, and provided the opportunity for the researchers to study them with genomic approaches, which had previously been impossible.
CENP-B, though not essential for natural chromosomes, has been assumed to be required for artificial centromere formation until now. A closely related protein, CENP-A, is actually the essential epigenetic marker for centromeres, and Black and his team have been able to direct the assembly of CENP-A onto the incoming HAC DNA.
The next-generation HACs made by Black and his team will allow for more thorough study of the essential components of functional chromosomes. Because a version of their HAC does not have the long repeating section, Black's team was able to use genomic approaches to analyze the sequence where centromeres formed. More reliable HACs will also open the door to complex synthetic biological systems that require longer sequences than can fit in viruses, the current common mode of delivering synthetic genetic systems.
This work was supported by the National Institutes of Health, the UPenn Cell and Molecular Biology Training Grant, the European Research Council, and the Wellcome Trust Senior Researcher Fellowship. The authors declare no conflict of interest.
Cell, Logsdon et al.: "Human Artificial Chromosomes that Bypass Centromeric DNA" https://www.cell.com/cell/fulltext/S0092-8674(19)30634-8
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