Richard Feynman’s dictum, “What I cannot create, I do not understand,” resonates with synthetic biologists, who seek to create artificial versions of natural living systems. One of these systems is the human chromosome. Although it is close to being “creatable”—in the sense synthetic biologists appreciate—it presents a central difficulty, the centromere.
Unlike the centromeres in baker’s yeast, the centromeres in human cells are not simply coded by a DNA sequence. They depend on a histone H3 variant, centromere protein A (CENP-A), to epigenetically specify their location. An added difficulty is that the DNA sequence of a natural human centromere incorporates thousands of repetitions of a 171-base-pair sequence—and such repetitive sequences are difficult to clone in the lab.
Quite the Gordian Knot. Confronted with this problem, scientists based at the University of Pennsylvania decided that they would, like Alexander the Great, implement a workaround solution. They biochemically delivered CENP-A directly to human artificial chromosome (HAC) DNA. Doing so greatly simplifies the building of HACs. For example, it avoids a supposedly unavoidable complication: the need for a high density of local CENP-B on naked DNA to facilitate assembly or stabilization of CENP-A nucleosomes.
CENP-B, though not essential for natural chromosomes, had been assumed to be required for artificial centromere formation.
Essentially, the University of Pennsylvania scientists, led by Ben Black, PhD, professor, forced CENP-A to associate with non-repetitive DNA sequences to form a new centromere for the HAC. The scientists described their approach in an article (“Human Artificial Chromosomes That Bypass Centromeric DNA”) that appeared July 25 in the journal Cell.
“Here, we improve HAC technology with a collection of HACs that include repetitive centromeric sequences or non-repetitive genomic sequences, testing each type for their dependence on seeding CENP-A nucleosome assembly,” the article’s authors wrote.
“Formed by an initial CENP-A nucleosome seeding strategy,” they continued, “a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types.”
In other words, Black and colleagues created two new HACs. Neither of them use CENP-B, and one is not repetitive.
“We wanted to see if we could break the rules by bestowing the DNA we put into the cell with epigenetic markers from the get go,” said Black. “We’ve taken our centromere bypass method to make a fully functional HAC without the cloning nightmares that repetitive centromere DNA has presented to mammalian chromosome engineers through the last two decades.”
“Building on our success,” he continued, “we and others in the synthetic chromosome field will now have a real chance to attain what has only been achieved so far in yeast cells.”
One of the next steps for this area of synthetic biology will be to link the Black lab’s centromere to sets of genes that others have designed. This step-by-step construction project is the goal of the Human Genome Project-Write, a collaboration to build that life-size synthetic chromosome. The Black lab’s contribution will help speed creating useful research and clinical tools based on synthetic chromosomes.
Going forward, HACs may be more reliably inherited in cell culture, and the regions where centromeres from may be subjected to genomic study, which had previously been impossible. 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.
“The centromere used to be called the black box of the chromosome,” Black declared. “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.”
“Think of the HACs we build now as model-sized chromosomes,” noted Glennis Logsdon, the current study’s first author and a doctoral student in Black’s lab at the time of the study. (He is now a postdoctoral fellow at the University of Washington.) “By being able to build a centromere on a HAC in a more straightforward way, we are closer to scaling up to full-size chromosomes.”