Biology subjects

Luke, B.

Publications and source records attributed to Luke, B..

12 recordsLinked to original sources

bioRxiv2024

Genome-wide conditional degron libraries for functional genomics

The yeast knockout library, comprised of strains carrying precise deletions of individual open reading frames (ORFs), has been a vital resource to understand gene function. However, accumulation of suppressor mutations in knockout strains can lead to erroneous functional annotations. Moreover, the library is limited to non-essential ORFs, and must be complemented with hypomorphic alleles of essential ORFs for genome- wide studies. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for conditional degradation of AID-tagged proteins. First, we determined that N-terminal tagging is at least twice more likely to inadvertently impair protein function across the proteome. We thus constructed two genome-wide libraries with over 5600 essential and non-essential proteins fused at the C-terminus with an AID tag and an optional fluorescent protein. Almost 90% of AID- tagged proteins were degraded in the presence of the auxin analog 5-Ph-IAA, with initial protein abundance and tag accessibility as limiting factors. Genome-wide screens for DNA damage response factors with the AID libraries revealed a role for the glucose signaling factor GSF2 in resistance to hydroxyurea, highlighting how these resources extend the toolbox for functional genomics in yeast.

systems biology↗

bioRxiv2024

MoCoLo: a testing framework for motif co-localization

Sequence-level data offers insights into biological processes through the interaction of two or more genomic features from the same or different molecular data types. Within motifs, this interaction is often explored via the co-occurrence of feature genomic tracks using fixed-segments or analytical tests that respectively require window size determination and risk of false positives from over-simplified models. Moreover, methods for robustly examining the co-localization of genomic features, and thereby understanding their spatial interaction, have been elusive. We present a new analytical method for examining feature interaction by introducing the notion of reciprocal co-occurrence, define statistics to estimate it, and hypotheses to test for it. Our approach leverages conditional motif co-occurrence events between features to infer their co-localization. Using reverse conditional probabilities and introducing a novel simulation approach that retains motif properties (e.g., length, guanine-content), our method further accounts for potential confounders in testing. As a proof-of-concept, MoCoLo confirmed the co-occurrence of histone markers in a breast cancer cell line. As a novel analysis, MoCoLo identified significant co-localization of oxidative DNA damage within non-B DNA forming regions that significantly differed between non-B DNA structures. Altogether, these findings demonstrate the potential utility of MoCoLo for testing spatial interactions between genomic features via their co-localization.

bioinformatics↗

bioRxiv2022

TERRA increases at short telomeres in yeast survivors and regulates survivor associated senescence (SAS)

Cancer cells achieve immortality by employing either homology-directed repair (HDR) or the telomerase enzyme to maintain telomeres. ALT (alternative lengthening of telomeres) refers to the subset of cancer cells that employ HDR. ALT mechanisms are strongly conserved from yeast to human cells, with the yeast equivalent being referred to as survivors. The non-coding RNA, TERRA and its ability to form RNA-DNA hybrids have been implicated in ALT/survivor maintenance by promoting HDR. It is not understood which telomeres in ALT/survivors engage in HDR, nor is it clear which telomeres upregulate TERRA. Using yeast survivors as a model for ALT, we demonstrate that HDR only occurs at telomeres when they become critically short. Moreover, TERRA levels steadily increase as telomeres shorten and decrease again following HDR-mediated recombination. Surprisingly, we observe that survivors undergo cycles of senescence, in a similar manner to non-survivors following telomerase loss, which we refer to as survivor associated senescence (SAS). Similar to "normal" senescence, we observe that RNA-DNA hybrids slow the rate of SAS by decreasing the rate of telomere shortening. In summary, TERRA RNA-DNA hybrids regulate telomere dysfunction-induced senescence in both pre- and post-crisis cells.

molecular biology↗

bioRxiv2022

Genetic requirements for repair of lesions caused by single genomic ribonucleotides in S phase

Single ribonucleoside monophosphates (rNMPs) are transiently present in eukaryotic genomes. The RNase H2-dependent ribonucleotide excision repair (RER) pathway ensures error-free genomic rNMP removal. In pathological conditions, genomic rNMP levels can rise and persist. If these rNMPs hydrolyse in, or prior to, S phase, toxic single-ended double-strand breaks (seDSBs) can occur upon an encounter with replication forks. How such rNMP-derived seDSB lesions are repaired is unclear. We employed a cell cycle phase restricted allele of RNase H2 as a genetic tool to induce nicks at rNMPs specifically in S phase to generate such lesions and study their repair. Here, we introduce a network of genes that maintain DNA integrity when rNMP-derived nick lesions arise during DNA replication. We use genetic methods to characterise the molecular requirements of a Top1-independent, rNMP-derived nick lesion repair (NLR). In NLR, the RAD52 epistasis group becomes essential for homology-directed repair (HDR). Moreover, the previously described Rtt101Mms1-Mms22 dependent ubiquitylation of histone H3 is essential for NLR in cells with high rNMP load, and loss of Rtt101Mms1-Mms22 combined with RNase H2 dysfunction leads to compromised cellular fitness. We discuss the genetic NLR network in the context of human disease, where cancer therapies may be able to exploit these synthetic lethalities.

genetics↗

bioRxiv2022

Epitope Mapping of SARS-CoV-2 Spike Protein Reveals Distinguishable Antibody Binding Activity of Vaccinated and Infected Individuals.

Previous studies have attempted to characterize the antibody response of individuals to the SARS-CoV-2 virus on a linear peptide level by utilizing peptide microarrays. These studies have helped to identify epitopes that have potential to be used for diagnostic tests to identify infected individuals, however, the immunological responses of individuals who have received the currently available Moderna mRNA-1273 or Pfizer BNT162b2 mRNA vaccines have not been characterized. We aimed to identify linear peptides of the SARS-CoV-2 spike protein that elicited high IgG or IgA binding activity and to compare the immunoreactivity of infected individuals to those who received both doses of either vaccines by utilizing peptide microarrays. Our results revealed peptide epitopes of significant IgG binding among recently infected individuals. Some of these peptides are located near functional domains implicated in the high infectivity of SARS-CoV-2. Vaccinated individuals lacked these distinct markers despite overall binding activity being similar.

immunology↗

bioRxiv2022

CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature of KRAS mutant cancers

Oncogenic RAS induces perinuclear translocation of the effector kinases ERK and CK2 and their scaffold, KSR1, forming endosomal signaling hubs termed perinuclear signaling centers (PSCs). PSCs are present in all cancer cell lines and tissues examined, suggesting that subcellular compartmentalization of oncogenic kinases drives tumorigenesis. However, the mechanism of perinuclear targeting, whether this location affects kinase substrate specificity, and the importance of PSCs in cancer are unclear. Here we show that the endosomal adaptor, TOLLIP, specifically tethers RAB11A+ signaling endosomes containing CK2 and KSR1 to the perinuclear ER. A predicted {beta}-hairpin fold in TOLLIP mediates binding to the KSR1 CA5 pseudo-kinase domain, recruiting CK2/KSR1 complexes to perinuclear endosomes. TOLLIP is essential for proliferation/survival of tumor cells carrying KRAS and NRAS mutations but not HRAS, BRAF, ERBB or PTEN lesions, or non-transformed cells. KRasG12D-induced lung lesions in Tollip-/- mice displayed reduced numbers of carcinomatous lesions, implicating TOLLIP in malignant progression. TOLLIP-dependent perinuclear CK2 was shown to phosphorylate discrete substrates, including proteins involved in translation and ribosome biogenesis such as RIOK1. Thus, TOLLIP is a key RAS pathway signaling adaptor in K/NRAS tumors whose inhibition is a specific vulnerability of these cancers.

cancer biology↗

bioRxiv2022

3'UTR-directed, kinase proximal mRNA decay inhibits C/EBPβ phosphorylation/activation to suppress senescence in tumor cells

C/EBP{beta} is a potent regulator of oncogene-induced senescence (OIS) and the SASP. C/EBP{beta} is post-translationally activated in OIS cells by the effector kinases ERK1/2 and CK2. However, in tumor cells C/EBP{beta} activation is suppressed by its 3UTR. 3'UTR regulation of protein activity (UPA) requires a G/U-rich element (GRE) and its cognate binding protein, HuR. These components segregate CEBPB transcripts away from a perinuclear compartment harboring ERK1/2 and CK2, restricting C/EBP{beta} from its activating kinases. We report here that the mRNA decay proteins UPF1 and Staufen1/2 are essential UPA factors enriched within the perinuclear cytoplasm. STAU1/2 and UPF1 overlap with CK2 on perinuclear signaling endosomes where they promote localized CEBPB mRNA decay. UPF1 or STAU1/2 depletion in tumor cells increased CEBPB transcripts adjacent to CK2 foci, coinciding with C/EBP{beta} activation and senescence. The GRE and an adjacent STAU binding site independently suppress C/EBP{beta}-mediated senescence, while a distinct 3UTR region inhibits its SASP-inducing activity. KrasG12D-driven lung tumors in mice carrying a Cebpb GRE deletion rarely progressed to malignant adenocarcinomas, demonstrating the importance of UPA to enable tumor progression in vivo. Thus, kinase-proximal mRNA decay is a novel mechanism that inhibits C/EBP{beta} activation in tumor cells to facilitate senescence bypass.

cancer biology↗

bioRxiv2020

Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 16,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 385,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 6 specific genes, and by clonal expansion. The clones in which a provirus integrated in an oncogene contributed to the survival of the clone comprise only a small fraction of the clones that persist in HIV-infected individuals on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist. Author SummaryIn HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists on ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as has been previously reported, integration favors highly-expressed genes. However, over time the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of six genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

microbiology↗

bioRxiv2020

Tumor IsomiR Encyclopedia (TIE): a pan-cancer database of miRNA isoforms

MicroRNAs (miRNAs) function as master regulators of gene expression in many physiological and pathological conditions including cancer. Sequence variants or isoforms (isomiRs) can account for between 40 to 60% of total miRNA counts, yet despite this overwhelming abundance, their function continues to be debated. Recent studies demonstrate that certain isomiRs can regulate unique sets of target mRNAs by altering their seed sequence or stabilizing 3 pairing, while others are decay intermediates indicating an active miRNA turnover. Given their short sequence length and high heterogeneity, mapping isomiRs can be challenging; without adequate depth and data aggregation, low frequency events are often disregarded. To address these challenges, we present the Tumor IsomiR Encyclopedia (TIE): a dynamic database of isomiRs from over 10,000 adult and pediatric tumor samples in The Cancer Genome Atlas (TCGA) and The Therapeutically Applicable Research to Generate Effective Treatments (TARGET) projects. A key novelty of TIE is its ability to annotate heterogeneous isomiR sequences and aggregate the variants obtained across all samples and datasets. The database provides annotation of templated and non-templated nucleotides as well as other advanced analysis. All data can be browsed online or downloaded as simple spreadsheets. Here we show analysis of isomiRs of miR-21 and miR-30a to demonstrate the utility of TIE. TIE search engine and data are hosted at https://isomir.ccr.cancer.gov/.

genomics↗

bioRxiv2020

Chromatin modifiers and recombination factors promote a telomere fold-back structure, that is lost during replicative senescence

Telomeres adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomeric chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence. Author summaryTelomeres are the protective caps of chromosome ends and prevent the activation of a local DNA damage response. In many organisms, telomeres engage in a loop-like structure which may provide an additional layer of end protection. As we still lack insight into the regulation of the folded telomere structure, we used budding yeast to establish a method to measure telomere folding and then study the genetic requirements for its establishment. We found that cells require the homologous recombination machinery as well as components of the DNA damage checkpoint to successfully establish a folded telomere. Through the deletion of telomerase in budding yeast, we investigated how telomere folding was regulated during replicative senescence, a process that occurs in the majority of telomerase negative human cells. During senescence, telomeres gradually shorten and erode until cells stop dividing which is a potent tumor suppressor and prevents unscheduled growth of potential cancer cells. We found, that the folded telomere structure is compromised as part of the cellular senescence response, but not due to telomere shortening per se. We think, that an altered telomeric chromatin environment during senescence is important to maintain an open state - which may be important for signaling or for repair.

genetics↗

bioRxiv2019

RNase H1 and H2 are differentially regulated to eliminate RNA-DNA hybrids

RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes, RNase H1 and H2, contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we have focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 is regulated in a strict cell cycle dependent manner, both in terms of its R-loop removal, and ribonucleotide excision repair functions. RNase H1, however, can function independent of cell cycle stage to remove R-loops, but appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.

molecular biology↗

bioRxiv2018

Checkpoint adaptation in repair-deficient cells drives aneuploidy and resistance to genotoxic agents

Human cancers frequently harbour mutations in DNA repair genes, rendering the use of DNA damaging agents as an effective therapeutic intervention. As therapy-resistant cells often arise, it is important to better understand the molecular pathways that drive resistance in order to facilitate the eventual targeting of such processes. We employ repair-defective diploid yeast as a model to demonstrate that, in response to genotoxic challenges, nearly all cells eventually undergo checkpoint adaptation, resulting in the generation of aneuploid cells with whole chromosome losses that have acquired resistance to the initial genotoxic challenge. We demonstrate that adaptation inhibition, either pharmacologically, or genetically, drastically reduces the occurrence of resistant cells. Additionally, the aneuploid phenotypes of the resistant cells can be specifically targeted to induce cytotoxicity. We provide evidence that TORC1 inhibition with rapamycin, in combination with DNA damaging agents, can prevent both checkpoint adaptation and the continued growth of aneuploid resistant cells.

cell biology↗