Design principles to tailor Hsp104 therapeutics.

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off-target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics.

Efficient formation of single-copy human artificial chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Centromere innovations within a mouse species.

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying centromere protein-A (CENP-A) nucleosomes at the nexus of a satellite repeat that we identified and termed π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 mega-base pairs of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance promotes accumulation of microtubule-binding components of the kinetochore and a microtubule-destabilizing kinesin of the inner centromere. We propose that the balance of pro- and anti-microtubule binding by the new centromere is what permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

Efficient Formation of Single-copy Human Artificial Chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125 bp DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. Here, we describe an approach that efficiently forms single-copy HACs. It employs a ~750 kb construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Centromere Innovations Within a Mouse Species.

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying CENP-A nucleosomes at the nexus of a satellite repeat that we identified and term π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 Mbp of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance drives accumulation of microtubule-binding components of the kinetochore, as well as a microtubule-destabilizing kinesin of the inner centromere. The balance of pro- and anti-microtubule-binding by the new centromere permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub.

Functional tags are ubiquitous in cell biology, and for studies of one chromosomal locus, the centromere, tags have been remarkably useful. The centromere directs chromosome inheritance at cell division. The location of the centromere is defined by a histone H3 variant, CENP-A. The regulation of the chromatin assembly pathway essential for centromere inheritance and function includes posttranslational modification (PTM) of key components, including CENP-A itself. Others have recently called into question the use of functional tags, with the claim that at least two widely used tags obscured the essentiality of one particular PTM, CENP-AK124 ubiquitination (ub). Here, we employ three independent gene replacement strategies that eliminate large, lysine-containing tags to interrogate these claims. Using these approaches, we find no evidence to support an essential function of CENP-AK124ub. Our general methodology will be useful to validate discoveries permitted by powerful functional tagging schemes at the centromere and other cellular locations.

The unique kind of human artificial chromosome: Bypassing the requirement for repetitive centromere DNA.

Centromeres are essential components of all eukaryotic chromosomes, including artificial/synthetic ones built in the laboratory. In humans, centromeres are typically located on repetitive α-satellite DNA, and these sequences are the "major ingredient" in first-generation human artificial chromosomes (HACs). Repetitive centromeric sequences present a major challenge for the design of synthetic mammalian chromosomes because they are difficult to synthesize, assemble, and characterize. Additionally, in most eukaryotes, centromeres are defined epigenetically. Here, we review the role of the genetic and epigenetic contributions to establishing centromere identity, highlighting recent work to hijack the epigenetic machinery to initiate centromere identity on a new generation of HACs built without α-satellite DNA. We also discuss the opportunities and challenges in developing useful unique sequence-based HACs.

Human Artificial Chromosomes that Bypass Centromeric DNA.

Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere-the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, 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. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.

The nucleosomes that mark centromere location on chromosomes old and new.

Proper segregation of chromosomes is an essential component of cell division. The centromere is the locus at which the kinetochore-the proteinaceous complex that ties chromosomes to microtubules-forms during mitosis and meiosis. Thus, the centromere is critical for equal segregation of chromosomes. The centromere is characterized by both protein and DNA elements: the histone H3 variant CENP-A epigenetically defines the location of the centromere while centromeric DNA sequences are neither necessary nor sufficient for centromere function. Paradoxically, the DNA sequences play a critical role in new centromere formation. In this essay, we discuss the contribution of both epigenetics and genetics at the centromere. Understanding these contributions is vital to efforts to control centromere formation on synthetic/artificial chromosomes and centromere strength on natural ones.