A Yeast-Based Screening System for Differential Identification of Poison Inhibitors and Catalytic Inhibitors of Human Topoisomerase I

Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern) -induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1 covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poison inhibitors developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. The aim of this study is to develop a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. This yeast-based screening system can identify human TOP1 poison inhibitors for anticancer efficacy as well as catalytic inhibitors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. In addition to distinguishing between such TOP1 catalytic inhibitors and TOP1 poison inhibitors, results from this yeast-based screening system will also allow elimination of compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.

An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain

As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.

A critical role of PvFtsH2 in the degradation of photodamaged D1 protein in common bean

Light is required for initiating chloroplast biogenesis and photosynthesis; however, the photosystem II reaction center (PSII RC) can be photodamaged. In this study, we characterized pvsl1, a seedling-lethal mutant of Phaseolus vulgaris. This mutant showed lethality when exposed to sunlight irradiation and a yellow-green leaf phenotype when grown in a growth chamber under low-light conditions. We developed 124 insertion/deletion (INDEL) markers based on resequencing data of Dalong1 and PI60234, two local Chinese common bean cultivars, for genetic mapping. We identified Phvul.002G190900, which encodes the PvFtsH2 protein, as the candidate gene for this pvsl1 mutation through fine-mapping and functional analysis. A single-base deletion occurred in the coding region of Phvul.002G190900 in the pvsl1 mutant, resulting in a frameshift mutation and a truncated protein lacking the Zn2+ metalloprotease domain. Suppressed expression of Phvul.002G190900 at the transcriptional level was detected, while no change in the subcellular localization signal was observed. The seedlings of pvsl1 exhibited hypersensitivity to photoinhibition stress. In the pvsl1 mutant, abnormal accumulation of the D1 protein indicated a failure to rapidly degrade damaged D1 protein in the PSII RC. The results of this study demonstrated that PvFtsH2 is critically required for survival and maintaining photosynthetic activity by degrading photodamaged PSII RC D1 protein in common bean.

Plastid caseinolytic protease OsClpR1 regulates chloroplast development and chloroplast RNA editing in rice

Background Plant plastidic caseinolytic protease (Clp) is a central part of the plastid protease network and consists of multiple subunits. The molecular functions of many Clps in plants, especially in crops, are not well known. Results In this study, we identified an albino lethal mutant al3 in rice, which produces albino leaves and dies at the seedling stage. Molecular cloning revealed that AL3 encodes a plastid caseinolytic protease, OsClpR1, homologous to Arabidopsis ClpR1 and is targeted to the chloroplast. Compared with the wild type, chloroplast structure in the al3 mutant was poorly developed. OsClpR1 was constitutively expressed in all rice tissues, especially in young leaves. The OsClpR1 mutation affected the transcript levels of chlorophyll biosynthesis and chloroplast development-related genes. The RNA editing efficiency of three chloroplast genes (rpl2, ndhB, ndhA) was remarkably reduced in al3. Using a yeast two-hybrid screen, we found that OsClpR1 interacted with OsClpP4, OsClpP5, OsClpP2, and OsClpS1. Conclusions Collectively, our results provide novel insights into the function of Clps in rice.

Fine Mapping and Identification of BnaC06.FtsH1, a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.

Validation of Omega Subunit of RNA Polymerase as a Functional Entity

The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ’ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β’, and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ’ subassembly. We observed that the interaction between α2ββ’ and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code’s position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ’. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β’ subunit, and it is the same for both types of mutants.

Maize pentatricopeptide repeat protein DEK41 affects cis-splicing of mitochondrial nad4 intron 3 and is required for normal seed development

The splicing of organelle-encoded mRNA in plants requires proteins encoded in the nucleus. The mechanism of splicing and the factors involved are not well understood. Pentatricopeptide repeat (PPR) proteins are known to participate in such RNA–protein interactions. Maize defective kernel 41 (dek41) is a seedling-lethal mutant that causes developmental defects. In this study, the Dek41 gene was cloned by Mutator tag isolation and allelic confirmation, and was found to encode a P-type PPR protein that targets mitochondria. Analysis of the mitochondrial RNA transcript profile revealed that dek41 mutations cause reduced splicing efficiency of mitochondrial nad4 intron 3. Immature dek41 kernels exhibited severe reductions in complex I assembly and NADH dehydrogenase activity. Up-regulated expression of alternative oxidase genes and deformed inner cristae of mitochondria in dek41, as revealed by TEM, indicated that proper splicing of nad4 is essential for correct mitochondrial functioning and morphology. Consistent with this finding, differentially expressed genes in the dek41 endosperm included those related to mitochondrial function and activity. Our results indicate that DEK41 is a PPR protein that affects cis-splicing of mitochondrial nad4 intron 3 and is required for correct mitochondrial functioning and maize kernel development.

Single-Nucleotide Human Disease Mutation Inactivates a Blood-Regenerative Enhancer

Coding and regulatory GATA2 mutations that deregulate protein expression and/or function cause MDS/AML (McReynolds et al., 2018). In the mouse, decreased GATA-2 expression impairs hematopoietic stem/progenitor cell (HSPC) genesis and function. While prior studies demonstrated the requirement for Gata2 +9.5 and -77 enhancers to regulate GATA-2 and HSPC transitions in vivo (Gao et al., 2013; Johnson et al., 2012; Johnson et al., 2015; Mehta et al., 2017; Sanalkumar et al., 2014), nothing is known about the contribution of the individual motifs within these enhancers for GATA-2 expression, hematopoiesis and hematopoietic regeneration. To elucidate mechanisms conferring +9.5 enhancer activity that regulate HSPC genesis and function, we used CRISPR/Cas9 technology to generate cis-element-mutant mouse strains. The first strain recapitulates human disease mutations and involves corruption of the E-box and a C>T transition within an Ets site (+9.5(E-box;Ets)), while retaining a GATA motif. The second strain contains the Ets 1017+572C>T transition (+9.5(Ets)), representing the most frequent noncoding GATA2 mutation in human patients (Ganapathi et al., 2015; Hsu et al., 2013). We compared the phenotypes of these strains with our previously generated +9.5(E-box;GATA)-/- strain (+9.5-/-) (Gao et al., 2013; Johnson et al., 2012) to ascertain how the +9.5 regulates hematopoietic processes. +9.5-/- embryos have nearly complete ablation of HSC emergence and die by E14. +9.5(E-box;Ets-/- embryos recapitulate +9.5 defects, indicating that neither the GATA nor Ets motifs suffice for +9.5 developmental functions. In contrast, +9.5(Ets)-/- mutant animals are born at normal Mendelian ratios - the sole example of a +9.5 mutation in the mouse that is not embryonic lethal. Mutant E15.5 embryos have minor decreases in fetal liver cell number (20%, P=0.023), no hemorrhaging or edema, and Gata2 mRNA is reduced 25-50% (P=0.006). +9.5(Ets)-/- adult mice exhibited normal steady-state hematopoiesis. To test if stress-dependent hematopoietic regeneration revealed Ets motif activity, 5-fluorouracil (5FU) was used to kill cycling cells and promote quiescent HSC proliferation. Two doses of 5FU (250 mg/kg) were administered at an interval of eleven days to stimulate maximal HSPC expansion (Xu et al., 2017). +9.5(Ets)-/- mice had a significantly reduced (P<0.0001) median survival of 14.5 days, versus 22 and 21 days for wild type (WT) and +9.5(Ets)+/-, respectively. We hypothesized that the hypersensitivity of +9.5(Ets)-/- mice to myeloablative stress was due to impaired HSC mobilization and/or recovery. To identify cell populations altered in +9.5(Ets)-/- bone marrow, we quantified immunophenotypic HSPCs in untreated and treated (9 and 11 days post-5FU) mice. In untreated mice, +9.5(Ets)-/- and WT HSC (Lin−Sca1+Kit+CD48-CD150+) numbers were similar. MPPs (Lin-Sca1+Kit+CD48-CD150-) were 9-fold lower in mutants. Nine and eleven days post-5FU treatment, HSCs increased 17- and 10-fold in WT (P<0.001 and P=0.003, respectively), but not +9.5(Ets)-/-, mice. To assess HSPC function, bone marrow was competitively transplanted into lethally-irradiated mice. Four months post-transplant, +9.5(Ets)-/- marrow was defective in multi-lineage repopulation (3-fold decrease in donor-derived cells from peripheral blood (P=0.0005), with significant reductions of HSPCs in bone marrow). These results demonstrate that the Ets motif confers Gata2 function as a key step in HSC regeneration. In summary, multiple cis-elements confer the developmental function of an essential enhancer, while a single cis-element mutated in disease confers regeneration in response to injury. Our results establish a paradigm in which the cis-element predisposition mutation, combined with a secondary insult, underlies pathogenesis and illustrates the power of micro-dissecting enhancers to unveil physiological and pathological mechanisms. Studies are underway to rigorously analyze the functional consequences of diverse secondary insults to identify conditions that cause bone marrow failure and hematologic malignancy. Comparing and contrasting developmental and regenerative genetic networks, including those operational in single cells from our unique models, will reveal broad principles and strategies to manipulate normal and malignant hematopoiesis, for example in the context of GATA-2-dependent pathologies. Disclosures No relevant conflicts of interest to declare.