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1. Structural basis for the preservation of a subset of topologically associating domains in interphase chromosomes upon cohesin depletion

   https://doi.org/10.7554/eLife.88564  

   Jeong, Davin; Shi, Guang; Li, Xin; Thirumalai, D (March 2024, eLife)

   Compartment formation in interphase chromosomes is a result of spatial segregation between euchromatin and heterochromatin on a few megabase pairs (Mbp) scale. On the sub-Mbp scales, topologically associating domains (TADs) appear as interacting domains along the diagonal in the ensemble averaged Hi-C contact map. Hi-C experiments showed that most of the TADs vanish upon deleting cohesin, while the compartment structure is maintained, and perhaps even enhanced. However, closer inspection of the data reveals that a non-negligible fraction of TADs is preserved (P-TADs) after cohesin loss. Imaging experiments show that, at the single-cell level, TAD-like structures are presenteven without cohesin. To provide a structural basis for these findings, we first used polymer simulations to show that certain TADs with epigenetic switches across their boundaries survive after depletion of loops. More importantly, the three-dimensional structures show that many of the P-TADs have sharp physical boundaries. Informed by the simulations, we analyzed the Hi-C maps (with and without cohesin) in mouse liver and human colorectal carcinoma cell lines, which affirmed that epigenetic switches and physical boundaries (calculated using the predicted 3D structures using the data-driven HIPPS method that uses Hi-C as the input) explain the origin of the P-TADs. Single-cell structures display TAD-like features in the absence of cohesin that are remarkably similar to the findings in imaging experiments. Some P-TADs, with physical boundaries, are relevant to the retention of enhancer–promoter/promoter–promoter interactions. Overall, our study shows that preservation of a subset of TADs upon removing cohesin is a robust phenomenon that is valid across multiple cell lines. 

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2. LKB1-Dependent Regulation of TPI1 Creates a Divergent Metabolic Liability between Human and Mouse Lung Adenocarcinoma

   https://doi.org/10.1158/2159-8290.CD-22-0805  

   Stein, Benjamin D.; Ferrarone, John R.; Gardner, Eric E.; Chang, Jae Won; Wu, David; Hollstein, Pablo E.; Liang, Roger J.; Yuan, Min; Chen, Qiuying; Coukos, John S.; et al (January 2023, Cancer Discovery)

   Abstract KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD), and activating mutations frequently co-occur with loss-of-function mutations in TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered comutation is highly aggressive in mouse lung adenocarcinoma (mLUAD). Here, we provide a mechanistic explanation for this difference by uncovering an evolutionary divergence in the regulation of triosephosphate isomerase (TPI1). In hLUAD, TPI1 activity is regulated via phosphorylation at Ser21 by the salt inducible kinases (SIK) in an LKB1-dependent manner, modulating flux between the completion of glycolysis and production of glycerol lipids. In mice, Ser21 of TPI1 is a Cys residue that can be oxidized to alter TPI1 activity without a need for SIKs or LKB1. Our findings suggest this metabolic flexibility is critical in rapidly growing cells with KRAS and TP53 mutations, explaining why the loss of LKB1 creates a liability in these tumors. Significance:Utilizing phosphoproteomics and metabolomics in genetically engineered human cell lines and genetically engineered mouse models (GEMM), we uncover an evolutionary divergence in metabolic regulation within a clinically relevant genotype of human LUAD with therapeutic implications. Our data provide a cautionary example of the limits of GEMMs as tools to study human diseases such as cancers.This article is highlighted in the In This Issue feature, p. 799 

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3. Differential requirements for the CENP-O complex reveal parallel PLK1 kinetochore recruitment pathways

   https://doi.org/10.1091/mbc.E20-11-0751  

   Nguyen, Alexandra L.; Fadel, Marie Diane; Cheeseman, Iain M. (April 2021, Molecular Biology of the Cell)

   Bloom, Kerry (Ed.)

   Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome-wide screens have identified a number of proteins that exhibit cell line–specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the four-subunit centromere-localized CENP-O complex, whose precise function has been difficult to define. Our results support a model in which the CENP-O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell-state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease. 

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4. Cell type-specific translational regulation by human DUS enzymes

   https://doi.org/10.1101/2023.11.03.565399  

   Yu, Nathan J; Dai, Wei; Li, Ang; He, Muhan; Kleiner, Ralph E (November 2023, bioRxiv)

   ABSTRACT Dihydrouridine is an abundant and conserved modified nucleoside present on tRNA, but characterization and functional studies of modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as an activity-based probe for human DUS enzymes. We map D modifications using RNA-protein crosslinking and chemical transformation and mutational profiling to reveal D modification sites on human tRNAs. Further, we knock out individual DUS genes in two human cell lines to investigate regulation of tRNA expression levels and codon-specific translation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation. 

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5. A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments

   https://doi.org/10.1038/s41467-022-28045-w  

   Zhao, Boyang; Rao, Yiyun; Leighow, Scott; O’Brien, Edward P.; Gilbert, Luke; Pritchard, Justin R. (February 2022, Nature Communications)

   Abstract A genetic knockout can be lethal to one human cell type while increasing growth rate in another. This context specificity confounds genetic analysis and prevents reproducible genome engineering. Genome-wide CRISPR compendia across most common human cell lines offer the largest opportunity to understand the biology of cell specificity. The prevailing viewpoint, synthetic lethality, occurs when a genetic alteration creates a unique CRISPR dependency. Here, we use machine learning for an unbiased investigation of cell type specificity. Quantifying model accuracy, we find that most cell type specific phenotypes are predicted by the function of related genes of wild-type sequence, not synthetic lethal relationships. These models then identify unexpected sets of 100-300 genes where reduced CRISPR measurements can produce genome-scale loss-of-function predictions across >18,000 genes. Thus, it is possible to reduce in vitro CRISPR libraries by orders of magnitude—with some information loss—when we remove redundant genes and not redundant sgRNAs. 

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