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name:Boeke

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Genomic context sensitizes regulatory elements to genetic disruption

Ordoñez, Raquel; Zhang, Weimin; Ellis, Gwen; Zhu, Yinan; Ashe, Hannah J; Ribeiro-Dos-Santos, André M; Brosh, Ran; Huang, Emily; Hogan, Megan S; Boeke, Jef D; Maurano, Matthew T
Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis. Thus it remains unclear to what extent enhancer function at native loci relies on surrounding genomic context. Using the Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the murine Igf2/H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2/H19 locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting the activity of payloads delivered to the endogenous locus or to a safe harbor locus (Hprt) revealed that the Igf2/H19 locus includes additional, previously unknown long-range regulatory elements. Exchanging components of the Igf2/H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster functioned poorly out of context, and required its native surroundings to activate Sox2 expression. Conversely, the Sox2 locus control region (LCR) could activate both Igf2 and H19 outside its native context, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across different cell types revealed that, while the H19 enhancers are tightly coordinated within their native locus, the Sox2 LCR acts more independently. We show that these enhancer clusters typify broader classes of loci genome-wide. Our results show that unexpected dependencies may influence even the most studied functional elements, and our synthetic regulatory genomics approach permits large-scale manipulation of complete loci to investigate the relationship between locus architecture and function.
PMCID:10541140
PMID: 37781588
CID: 5606642

Gene loss and cis-regulatory novelty shaped core histone gene evolution in the apiculate yeast Hanseniaspora uvarum

Haase, Max A B; Steenwyk, Jacob L; Boeke, Jef D
Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation - mediated by the trans-regulator Spt10 - is ancient, likely emerging between 320-380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage (FEL), which coincided with the loss of one copy of its paralogous core histones genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora FEL.
PMID: 38271560
ISSN: 1943-2631
CID: 5625232

Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae

Cheng, Li; Zhao, Shijun; Li, Tianyi; Hou, Sha; Luo, Zhouqing; Xu, Jinsheng; Yu, Wenfei; Jiang, Shuangying; Monti, Marco; Schindler, Daniel; Zhang, Weimin; Hou, Chunhui; Ma, Yingxin; Cai, Yizhi; Boeke, Jef D; Dai, Junbiao
Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae.
PMCID:10817965
PMID: 38278805
ISSN: 2041-1723
CID: 5625492

Author Correction: Longitudinal scRNA-seq analysis in mouse and human informs optimization of rapid mouse astrocyte differentiation protocols

Frazel, Paul W; Labib, David; Fisher, Theodore; Brosh, Ran; Pirjanian, Nicolette; Marchildon, Anne; Boeke, Jef D; Fossati, Valentina; Liddelow, Shane A
PMID: 37996532
ISSN: 1546-1726
CID: 5608832

Humanization reveals pervasive incompatibility of yeast and human kinetochore components

Ólafsson, Guðjón; Haase, Max A B; Boeke, Jef D
Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone-H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast) marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful, however co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast, however this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell-cycle progression and kinetochore function in wild-type cells. Suppressors of CENP-A toxicity included gene deletions of subunits of three conserved chromatin-remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.
PMID: 37962556
ISSN: 2160-1836
CID: 5610642

Super-enhancers include classical enhancers and facilitators to fully activate gene expression

Blayney, Joseph W; Francis, Helena; Rampasekova, Alexandra; Camellato, Brendan; Mitchell, Leslie; Stolper, Rosa; Cornell, Lucy; Babbs, Christian; Boeke, Jef D; Higgs, Douglas R; Kassouf, Mira
Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.
PMID: 38101409
ISSN: 1097-4172
CID: 5589022

Manipulating the 3D organization of the largest synthetic yeast chromosome

Zhang, Weimin; Lazar-Stefanita, Luciana; Yamashita, Hitoyoshi; Shen, Michael J; Mitchell, Leslie A; Kurasawa, Hikaru; Lobzaev, Evgenii; Fanfani, Viola; Haase, Max A B; Sun, Xiaoji; Jiang, Qingwen; Goldberg, Gregory W; Ichikawa, David M; Lauer, Stephanie L; McCulloch, Laura H; Easo, Nicole; Lin, S Jiaming; Camellato, Brendan R; Zhu, Yinan; Cai, Jitong; Xu, Zhuwei; Zhao, Yu; Sacasa, Maya; ,; Noyes, Marcus B; Bader, Joel S; Deutsch, Samuel; Stracquadanio, Giovanni; Aizawa, Yasunori; Dai, Junbiao; Boeke, Jef D
Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.
PMID: 37944526
ISSN: 1097-4164
CID: 5612832

Building a eukaryotic chromosome arm by de novo design and synthesis

Jiang, Shuangying; Luo, Zhouqing; Wu, Jie; Yu, Kang; Zhao, Shijun; Cai, Zelin; Yu, Wenfei; Wang, Hui; Cheng, Li; Liang, Zhenzhen; Gao, Hui; Monti, Marco; Schindler, Daniel; Huang, Linsen; Zeng, Cheng; Zhang, Weimin; Zhou, Chun; Tang, Yuanwei; Li, Tianyi; Ma, Yingxin; Cai, Yizhi; Boeke, Jef D; Zhao, Qiao; Dai, Junbiao
The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a "one-amino-acid-one-codon" strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.
PMCID:10689750
PMID: 38036514
ISSN: 2041-1723
CID: 5589872

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

Zhao, Yu; Coelho, Camila; Hughes, Amanda L; Lazar-Stefanita, Luciana; Yang, Sandy; Brooks, Aaron N; Walker, Roy S K; Zhang, Weimin; Lauer, Stephanie; Hernandez, Cindy; Cai, Jitong; Mitchell, Leslie A; Agmon, Neta; Shen, Yue; Sall, Joseph; Fanfani, Viola; Jalan, Anavi; Rivera, Jordan; Liang, Feng-Xia; Bader, Joel S; Stracquadanio, Giovanni; Steinmetz, Lars M; Cai, Yizhi; Boeke, Jef D
The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASer
PMID: 37944511
ISSN: 1097-4172
CID: 5590882

Design, construction, and functional characterization of a tRNA neochromosome in yeast

Schindler, Daniel; Walker, Roy S K; Jiang, Shuangying; Brooks, Aaron N; Wang, Yun; Müller, Carolin A; Cockram, Charlotte; Luo, Yisha; García, Alicia; Schraivogel, Daniel; Mozziconacci, Julien; Pena, Noah; Assari, Mahdi; Sánchez Olmos, María Del Carmen; Zhao, Yu; Ballerini, Alba; Blount, Benjamin A; Cai, Jitong; Ogunlana, Lois; Liu, Wei; Jönsson, Katarina; Abramczyk, Dariusz; Garcia-Ruiz, Eva; Turowski, Tomasz W; Swidah, Reem; Ellis, Tom; Pan, Tao; Antequera, Francisco; Shen, Yue; Nieduszynski, Conrad A; Koszul, Romain; Dai, Junbiao; Steinmetz, Lars M; Boeke, Jef D; Cai, Yizhi
Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
PMID: 37944512
ISSN: 1097-4172
CID: 5590892