Precision off-the-shelf natural killer cell therapies for oncology with logic-gated gene circuits.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis (5-year survival rate of 30.5% in the United States). Designing cell therapies to target AML is challenging because no single tumor-associated antigen (TAA) is highly expressed on all cancer subpopulations.

A Quantitative Genetic Interaction Map of HIV Infection.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways.

The Set1 complex is dimeric and acts with Jhd2 demethylation to convey symmetrical H3K4 trimethylation.

Epigenetic modifications can maintain or alter the inherent symmetry of the nucleosome. However, the mechanisms that deposit and/or propagate symmetry or asymmetry are not understood. Here we report that yeast Set1C/COMPASS (complex of proteins associated with Set1) is dimeric and, consequently, symmetrically trimethylates histone 3 Lys4 (H3K4me3) on promoter nucleosomes.

Protein Moonlighting Revealed by Noncatalytic Phenotypes of Yeast Enzymes.

A single gene can partake in several biological processes, and therefore gene deletions can lead to different-sometimes unexpected-phenotypes. However, it is not always clear whether such pleiotropy reflects the loss of a unique molecular activity involved in different processes or the loss of a multifunctional protein. Here, using metabolism as a model, we systematically test the null hypothesis that enzyme phenotypes depend on a single annotated molecular function, namely their catalysis.

DNA Preparation from .

This protocol includes two methods for genomic DNA preparation from The first is a quick method for preparation of DNA suitable for polymerase chain reaction (PCR) genotyping. The second, longer method yields high-quality DNA that can be used for amplification of targeting cassettes.

Genetic Interaction Score (S-Score) Calculation, Clustering, and Visualization of Genetic Interaction Profiles for Yeast.

This protocol describes computational analysis of genetic interaction screens, ranging from data capture (plate imaging) to downstream analyses. Plate imaging approaches using both digital camera and office flatbed scanners are included, along with a protocol for the extraction of colony size measurements from the resulting images. A commonly used genetic interaction scoring method, calculation of the S-score, is discussed.

High-Throughput Quantitative Genetic Interaction Mapping in the Fission Yeast .

Epistasis mapping, in which the phenotype that emerges from combining pairs of mutations is measured quantitatively, is a powerful tool for unbiased study of gene function. When performed at a large scale, this approach has been used to assign function to previously uncharacterized genes, define functional modules and pathways, and study their cross talk. These experiments rely heavily on methods for rapid sampling of binary combinations of mutant alleles by systematic generation of a series of double mutants.

Transformation of in a 96-Well Format.

This protocol describes chemical transformation of with linear DNA in a 96-well format. This procedure has been successfully used for large-scale strain construction in fission yeast.

Genetic Interaction Mapping in Using the Pombe Epistasis Mapper (PEM) System and a ROTOR HDA Colony Replicating Robot in a 1536 Array Format.

This protocol describes an optimized high-throughput procedure for generating double deletion mutants in using the colony replicating robot ROTOR HDA and the PEM (pombe epistasis mapper) system. The method is based on generating high-density colony arrays (1536 colonies per agar plate) and passaging them through a series of antidiploid and mating-type selection (ADS-MTS) and double-mutant selection (DMS) steps. Detailed program parameters for each individual replication step are provided.

Genetic interaction mapping in mammalian cells using CRISPR interference.

We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation.

Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision.

Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells.

We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and "protein-level dependency" studies-a systematic technique that uncovers post-transcriptional regulation-to delineate the network of factors that control the expression of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs.

Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast.

Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex.

Genetic Interaction Landscape Reveals Critical Requirements for Schizosaccharomyces pombe Brc1 in DNA Damage Response Mutants.

Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN).

Evolutionarily conserved genetic interactions with budding and fission yeast MutS identify orthologous relationships in mismatch repair-deficient cancer cells.

Background: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death.

Yeast X-chromosome-associated protein 5 (Xap5) functions with H2A.Z to suppress aberrant transcripts.

Chromatin regulatory proteins affect diverse developmental and environmental response pathways via their influence on nuclear processes such as the regulation of gene expression. Through a genome-wide genetic screen, we implicate a novel protein called X-chromosome-associated protein 5 (Xap5) in chromatin regulation. We show that Xap5 is a chromatin-associated protein acting in a similar manner as the histone variant H2A.

Identification of a BET family bromodomain/casein kinase II/TAF-containing complex as a regulator of mitotic condensin function.

Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function.

A Pil1-Sle1-Syj1-Tax4 functional pathway links eisosomes with PI(4,5)P2 regulation.

Stable compartments of the plasma membrane promote a wide range of cellular functions. In yeast cells, cytosolic structures called eisosomes generate prominent cortical invaginations of unknown function. Through a series of genetic screens in fission yeast, we found that the eisosome proteins Pil1 and Sle1 function with the synaptojanin-like lipid phosphatase Syj1 and its ligand Tax4.

Quantitative genetic-interaction mapping in mammalian cells.

Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed ∼11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map.

Replication fork collapse and genome instability in a deoxycytidylate deaminase mutant.

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.

Hho1p, the linker histone of Saccharomyces cerevisiae, is important for the proper chromatin organization in vivo.

Despite the existence of certain differences between yeast and higher eukaryotic cells a considerable part of our knowledge on chromatin structure and function has been obtained by experimenting on Saccharomyces cerevisiae. One of the peculiarities of S. cerevisiae cells is the unusual and less abundant linker histone, Hho1p.

Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation.