The unique kind of human artificial chromosome: Bypassing the requirement for repetitive centromere DNA
Introduction
Since the first de novo synthesis of a prokaryotic genome [1], the prospect of building eukaryotic genomes has enticed synthetic biologists. If we could build even a single chromosome, it would unlock numerous possibilities, including engineering new gene circuits or genes of any sort. If we could build an entire eukaryotic genome, one would be presented with the ultimate synthetic biology toolbox, which could lead to transformative advances such as transplantable human organs harvested from livestock animals and virus-resistant cell lines for faithful antibody generation [2]. Work in budding yeast reported within the last six years has broken ground by generating the first entirely synthetic yeast chromosomes, combining the de novo synthesis approaches used in the prokaryotic system with recombination-mediated assembly of small chromosome building blocks [[3], [4], [5], [6]]. Since budding yeast chromosomes are tiny compared to their mammalian counterparts (the entire haploid budding yeast genome is 12 Mbp, while the “puny” human chromosome 21 is ~48 Mbp, and the entire haploid human genome is estimated to be ~3100 Mbp), one might envision that the substantial “scale up” will be the greatest challenge to translating success from yeast to mammalian genomes. That could prove true, but advances in DNA synthesis/assembly methodologies and instrumentation are likely to continue shortening this particular gap. However, other major challenges include the inclusion of essential mammalian chromosomal elements, the delivery of large artificial chromosomes into mammalian cells, and the efficiency in isolating cells that carry a functional artificial chromosome.
Over the last twenty years, the human artificial chromosome (HAC) field has contributed a wealth of information to circumvent some of these potential issues by defining the minimal components required for HAC propagation and inheritance through cell divisions alongside their natural chromosome counterparts. While these efforts have been reviewed previously [7,8] and elsewhere in this issue, we briefly mention the current possible “parts list” for HACs: telomeres, an origin of replication, and a centromere (Fig. 1). Telomeres are genetically encoded repeats that cap the ends of natural chromosomes in mammals and budding yeast, and the addition of telomere sequences to the ends of a linear HAC molecule is sufficient to protect HAC DNA ends [9]. However, telomeres are only needed if there actually are DNA ends, so one can also generate circular HACs that obviate this requirement [10,11](Fig. 1). While strict sequence-dependent origins of replication are required in budding yeast (the classic autonomous replicating sequence; ARS [12]), origins fire from a variety of sequences in mammals. Some differences in efficiency exist depending on sequence [13], but it remains unclear if a lack of efficient origin firing ever compromises HAC formation or stability, as long as sufficient lengths of mammalian genomic DNA is present to represent possible origin firing locations. The final requirement, the centromere, poses a greater challenge. This review will discuss efforts to use HACs as a tool to resolve these problems. In particular, HACs made from a non-repetitive DNA template can alleviate many of the challenges caused by the repetitive DNA typically found at human centromeres.
Section snippets
What is the centromere, and why is centromere formation a challenge for HAC formation?
The centromere is the locus responsible for equal segregation of chromosomes during cell division [14]. During mitosis, the centromere directs both kinetochore formation and protects cohesion of sister chromatids until their synchronous separation at the onset of anaphase. The kinetochore is the proteinaceous complex that binds to the spindle microtubules that drive chromosome alignment and subsequent segregation to the daughter cells. Thus, centromere formation is a requirement for a synthetic
Why was repetitive DNA assumed to be a requirement for centromere formation?
While centromeres are defined epigenetically in most eukaryotes, many mammals, including humans and mice, harbor repetitive DNA sequences at their centromeres [33]. In humans, these sequences are known as α-satellite DNA. α-satellite DNA consists of 171 bp repeats that are >60% identical to one another and typically organized in higher order repeats (HORs) of ~0.3–6 kb [[34], [35], [36]]. Despite being neither necessary nor sufficient for specifying centromere location, they underlie chromatin
Can CENP-B and α-satellite DNA be bypassed during HAC formation?
While CENP-B boxes and α-satellite DNA have been shown to contribute to centromere formation, numerous instances in nature exist in which animals have functional centromeres on non-repetitive DNA. In humans, there are many reported instances of neocentromeres, in which a centromere has formed at a new chromosomal location devoid of centromeric repeats or enrichment sites for CENP-B [45,46]. The precise mechanism by which neocentromeres form is not understood. Experimental neocentromeres, whose
DNA sequence matters!
A possible conclusion from this finding is that the only function of α-satellite DNA is the recruitment of CENP-B, but this appears to be far too simplistic. The α-satellite HACs are more efficiently formed through epigenetic centromere seeding, either in the absence or presence of CENP-B protein, than are the 4q21 HACs [67]. Thus, α-satellite seems especially “receptive” to epigenetic centromere seeding. On the other hand, α-satellite constructs with a low density of CENP-B boxes have never
Outlook for non-repetitive DNA-based HACs and mammalian synthetic chromosomes
Taking into account the present-state HACs, what are some of the important features beyond the basic functions driven by the minimal set of genetic and epigenetic elements all HACs require (Fig. 1)? We think the list of desired features should include: avoiding long stretches of repetitive centromere DNA, forming without needing to acquire genomic sequences from the host cell, avoiding multimerization that amplifies the copy number of the genetic payload, and avoiding rearrangements of the
CRediT authorship contribution statement
Craig W. Gambogi: Writing - original draft, Writing - review & editing. Jennine M. Dawicki-McKenna: Writing - review & editing. Glennis A. Logsdon: Writing - review & editing. Ben E. Black: Writing - original draft, Writing - review & editing.
Acknowledgements
The authors gratefully acknowledge support from NIH grants GM130302 (to B.E.B.) and GM134558 (to G.A.L.).
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