Faculty Bibliography

Human retrotransposon insertions are often associated with diseases. In the case of the neurodegenerative X-Linked Dystonia-Parkinsonism disease, a human-specific SINE-VNTR-Alu subfamily F retrotransposon was inserted in intron 32 of the TAF1 gene. Here, we genomically rewrote a portion of the mouse Taf1 allele with the corresponding 78-kb XDP patient derived TAF1 allele. In mESCs, the presence of the intronic SVAs-rather than the hybrid gene structure-reduces hyTAF1 levels. This leads to transcriptional downregulation of genes with TATA box enriched in their promoters and triggering apoptosis. Chromatin and transcriptome profiling revealed that intronic SVAs are actively transcribed, forming barriers that likely impede transcription elongation. In mice, neuronal lineage TAF1 humanization resulted lethality of male progeny within two months. XDP male mice had severe atrophy centered on the striatum-the same affected brain region in XDP patients. Lastly, CRISPRa-mediated activation of hyTAF1 restored mESC viability, suggesting boosting TAF1 transcription as a therapeutic approach.

Extrachromosomal DNA (ecDNA) drives oncogene amplification and intratumoural heterogeneity in aggressive cancers. While transposable element reactivation is common in cancer, its role on ecDNA remains unexplored. Here we map the 3D architecture of MYC-amplified ecDNA in colorectal cancer cells and identify 68 ecDNA-interacting elements-genomic loci enriched for transposable elements that are frequently integrated onto ecDNA. We focus on an L1M4a1#LINE/L1 fragment co-amplified with MYC, which functions only in the ecDNA-amplified context. Using CRISPR-CATCH, CRISPR interference and reporter assays, we confirm its presence on ecDNA, enhancer activity and essentiality for cancer cell fitness. These findings reveal that repetitive elements can be reactivated and co-opted as functional rather than inactive sequences on ecDNA, potentially driving oncogene expression and tumour evolution. Our study uncovers a mechanism by which ecDNA harnesses repetitive elements to shape cancer phenotypes, with implications for diagnosis and therapy.

Extrachromosomal DNA (ecDNA), a major focal oncogene amplification mode found across cancer, has recently regained attention as an emerging cancer hallmark, with a pervasive presence across cancers. With technical advancements such as high-coverage sequencing and live-cell genome imaging, we can now investigate the behaviors and functions of ecDNA. However, we still lack an understanding of how to eliminate ecDNA. We observed depletion of cells containing ecDNA during lentiviral but not transposon-based transduction while we sought to investigate the mechanism of ecDNA behavior. This discovery may provide critical information on utilizing a lentiviral system in emerging ecDNA research. Additionally, this observation suggests specific sensitivities for cells with ecDNA.

The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5' UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.

Extrachromosomal DNA (ecDNA) is a hallmark of aggressive cancer, contributing to both oncogene amplification and tumor heterogeneity. Here, we used Hi-C, super-resolution imaging, and long-read sequencing to explore the nuclear architecture of MYC-amplified ecDNA in colorectal cancer cells. Intriguingly, we observed frequent spatial proximity between ecDNA and 68 repetitive elements which we called ecDNA-interacting elements or EIEs. To characterize a potential regulatory role of EIEs, we focused on a fragment of the L1M4a1#LINE/L1 which we found to be co-amplified with MYC on ecDNA, gaining enhancer-associated chromatin marks in contrast to its normally silenced state. This EIE, in particular, existed as a naturally occurring structural variant upstream of MYC, gaining oncogenic potential in the transcriptionally permissive ecDNA environment. This EIE sequence is sufficient to enhance MYC expression and is required for cancer cell fitness. These findings suggest that silent repetitive genomic elements can be reactivated on ecDNA, leading to functional cooption and amplification. Repeat element activation on ecDNA represents a mechanism of accelerated evolution and tumor heterogeneity and may have diagnostic and therapeutic potential.

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.

In the era of synthetic biology, design, construction, and utilization of synthetic chromosomes with unique features provide a strategy to study complex cellular processes such as aging. Herein, we successfully construct the 884 Kb synXIII of Saccharomyces cerevisiae to investigate replicative aging using these synthetic strains. We verify that up-regulation of a rRNA-related transcriptional factor, RRN9, positively influence replicative lifespan. Using SCRaMbLE system that enables inducible whole-genome rearrangement on synXIII, we obtain 20 SCRaMbLEd synXIII strains with extended lifespan. Transcriptome analysis reveal the expression of genes involve in global protein synthesis is up-regulated in longer-lived strains. We establish causal links between genotypic change and the long-lived phenotype via reconstruction of some key structural variations observed in post-SCRaMbLE strains and further demonstrate combinatorial effects of multiple aging regulators on lifespan extension. Our findings underscore the potential of synthetic yeasts in unveiling the function of aging-related genes.

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects.

Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'^1-4. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.