Faculty Bibliography

Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway - even essential ones - while maintaining low false positive rates (less than 1 in 10⁸ per cell). This plasmid, dubbed "Superloser", was designed with reduced sequence similarity to commonly used yeast plasmids (i.e. pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker URA3 to reduce spontaneous 5-fluoroorotic acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof of concept shuffling experiment: swapping yeast's core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4×10⁹) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as low as 1 in 10⁸ cells.

The Origin Recognition Complex (ORC) is essential for replication, heterochromatin formation, telomere maintenance and genome stability in eukaryotes. Here we present the structure of the yeast Orc1 BAH domain bound to the nucleosome core particle. Our data reveal that Orc1, unlike its close homolog Sir3 involved in gene silencing, does not appear to discriminate between acetylated and non-acetylated lysine 16, modification states of the histone H4 tail that specify open and closed chromatin respectively. We elucidate the mechanism for this unique feature of Orc1 and hypothesize that its ability to interact with nucleosomes regardless of K16 modification state enables it to perform critical functions in both hetero- and euchromatin. We also show that direct interactions with nucleosomes are essential for Orc1 to maintain the integrity of rDNA borders during meiosis, a process distinct and independent from its known roles in silencing and replication.

Age-related Macular Degeneration (AMD) is a leading cause of blindness in the developed world, especially in aging populations, and is therefore an important target for new therapeutic development. Recently, there have been several studies demonstrating strong associations between AMD and sites of heritable genetic variation at multiple loci, including a highly significant association at 10q26. The 10q26 risk region contains two genes, HTRA1 and ARMS2, both of which have been separately implicated as causative for the disease, as well as dozens of sites of non-coding variation. To date, no studies have successfully pinpointed which of these variant sites are functional in AMD, nor definitively identified which genes in the region are targets of such regulatory variation. In order to efficiently decipher which sites are functional in AMD phenotypes, we describe a general framework for combinatorial assembly of large 'synthetic haplotypes' along with delivery to relevant disease cell types for downstream functional analysis. We demonstrate the successful and highly efficient assembly of a first-draft 119kb wild-type 'assemblon' covering the HTRA1/ARMS2 risk region. We further propose the parallelized assembly of a library of combinatorial variant synthetic haplotypes covering the region, delivery and analysis of which will identify functional sites and their effects, leading to an improved understanding of AMD development. We anticipate that the methodology proposed here is highly generalizable towards the difficult problem of identifying truly functional variants from those discovered via GWAS or other genetic association studies.

Retrotransposable elements are deleterious at many levels, and the failure of host surveillance systems for these elements can thus have negative consequences. However, the contribution of retrotransposon activity to ageing and age-associated diseases is not known. Here we show that during cellular senescence, L1 (also known as LINE-1) retrotransposable elements become transcriptionally derepressed and activate a type-I interferon (IFN-I) response. The IFN-I response is a phenotype of late senescence and contributes to the maintenance of the senescence-associated secretory phenotype. The IFN-I response is triggered by cytoplasmic L1 cDNA, and is antagonized by inhibitors of the L1 reverse transcriptase. Treatment of aged mice with the nucleoside reverse transcriptase inhibitor lamivudine downregulated IFN-I activation and age-associated inflammation (inflammaging) in several tissues. We propose that the activation of retrotransposons is an important component of sterile inflammation that is a hallmark of ageing, and that L1 reverse transcriptase is a relevant target for the treatment of age-associated disorders.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb human HPRT1 gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells

Background/UNASSIGNED:Transposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA. Results/UNASSIGNED:Here we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants), as well as somatically-acquired L1 insertions that distinguish cancer genomes from a patient's constitutional make-up. Conclusions/UNASSIGNED:TIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes.

Engineering multicellularity is one of the next breakthroughs for Synthetic Biology. A key bottleneck to building multicellular systems is the lack of a scalable signaling language with a large number of interfaces that can be used simultaneously. Here, we present a modular, scalable, intercellular signaling language in yeast based on fungal mating peptide/G-protein-coupled receptor (GPCR) pairs harnessed from nature. First, through genome-mining, we assemble 32 functional peptide-GPCR signaling interfaces with a range of dose-response characteristics. Next, we demonstrate that these interfaces can be combined into two-cell communication links, which serve as assembly units for higher-order communication topologies. Finally, we show 56 functional, two-cell links, which we use to assemble three- to six-member communication topologies and a three-member interdependent community. Importantly, our peptide-GPCR language is scalable and tunable by genetic encoding, requires minimal component engineering, and should be massively scalable by further application of our genome mining pipeline or directed evolution.

Quiescent cells exploit an array of transcription factors to activate stress response machinery and maintain survival under nutrient-limited conditions. Our recent findings reveal that these transcription factors also play an important role in the exit of quiescence and regrowth. By studying Saccharomyces cerevisiae under a continuous, nutrient-limited condition, we found that Msn2 and Msn4 function as master regulators of glycolytic genes in the quiescent-like phase. They control the timing of transition from quiescence to growth by regulating the accumulation rate of acetyl-CoA, a key metabolite that is downstream of glycolysis and drives growth. These findings suggest a model that Msn2/4 not only protect the cells from starvation but also facilitate their regrowth from quiescence. Thus, understanding the functions of stress response transcription factors in metabolic regulation will provide deeper insight into how quiescent cells manage the capacity of regrowth.

Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)^1,2. This is likely to reflect accidents of genome history, such as telomere-telomere fusions and genome duplication events^3-5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state⁶. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae^7-10, but CRISPR-Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR-Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome-chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change.

The LINE-1/L1 retrotransposon is a transposable element still active in the human genome. Most retrotransposons in the genome are inactive or repressed by several host mechanisms. In specific contexts, active L1 retrotransposons may evade repression and copy themselves into new genomic loci. Despite a general knowledge of the L1 life cycle, little was known about the dynamics of L1 proteins and function during the different stages of the host cell cycle. Our work highlighted a well-orchestrated localization of L1 proteins and mRNA that take advantage of mitotic nuclear membrane breakdown. Once in the nucleus, L1 ribonucleoproteins (RNPs) are able to retrotranspose during the S phase when L1 retrotransposition peaks. Our conclusions highlight previously unappreciated features of the L1 life cycle, such as the differences between cytoplasmic and nuclear RNPs and the cycling of L1 ORF1 protein and L1 activity during progression through the cell cycle. These new observations are discussed here in light of the evolutionary arms race between L1 retrotransposons and the host cell.