Initial Functional Characterization Research Articles

The evolutionarily conserved PhLP3 is essential for sperm development in Drosophila melanogaster.

Phosducin-like proteins (PhLP) are thioredoxin domain-containing proteins that are highly conserved across unicellular and multicellular organisms. PhLP family proteins are hypothesized to function as co-chaperones in the folding of cytoskeletal proteins. Here, we present the initial molecular, biochemical, and functional characterization of CG4511 as Drosophila melanogaster PhLP3. We cloned the gene into a bacterial expression vector and produced enzymatically active recombinant PhLP3, which showed similar kinetics to previously characterized orthologues. A fly strain homozygous for a P-element insertion in the 5' UTR of the PhLP3 gene exhibited significant downregulation of PhLP3 expression. We found these male flies to be sterile. Microscopic analysis revealed altered testes morphology and impairment of spermiogenesis, leading to a lack of mature sperm. Among the most significant observations was the lack of actin cones during sperm maturation. Excision of the P-element insertion in PhLP3 restored male fertility, spermiogenesis, and seminal vesicle size. Given the high level of conservation of PhLP3, our data suggests PhLP3 may be an important regulator of sperm development across species.

HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro.

Disease-associated microglia (DAM), initially described in mouse models of neurodegenerative diseases, have been classified into two related states; starting from a TREM2-independent DAM1 state to a TREM2 dependent state termed DAM2, with each state being characterized by the expression of specific marker genes1. Recently, single-cell (sc)RNA-Seq studies have reported the existence of DAMs in humans2-6; however, whether DAMs play beneficial or detrimental roles in the context of neurodegeneration is still under debate7,8. Here, we present a pharmacological approach to mimic human DAM in vitro by exposing different human microglia models to selected histone deacetylase (HDAC) inhibitors. We also provide an initial functional characterization of our model system, showing a specific increase of amyloid beta phagocytosis along with a reduction of MCP-1 secretion. Additionally, we report an increase in MITF expression, a transcription factor previously described to drive expression towards the DAM phenotype. We further identify CADM1, LIPA and SCIN as DAM-marker genes shared across various proposed DAM signatures and in our model systems. Overall, our strategy for targeted microglial polarization bears great potential to further explore human DAM function and biology.

Viroid and viroid-like elements in plants and plant-associated microbiota: a new layer of biodiversity for plant holobionts.

The functional relevance of plant-associated microorganisms is theoretically framed within the holobiont concept. The role of viruses in plant holobionts is being recognized both for their direct effects when hosted in plants (cryptic plant viruses) and for their indirect effects when infecting microorganisms associated with plants in tripartite interactions (e.g. mycoviruses and bacteriophages). We argue that viroids, the smallest infectious agents typically infecting only plant hosts, must also be included in plant holobiont studies. The same applies to the recently discovered large number of viroid-like elements infecting hosts of other life kingdoms that are closely associated with plants. Here we also describe in depth the diversity of such viroid-like elements and their initial functional characterization in plant-associated fungi.

Chemical characterization of refuse derived fuel (RDF) using Py-GC/MS

The conversion of municipal solid waste (MSW) to refuse-derived fuel (RDF) and its utilization as an alternative fuel in the cement industry has been an emerging trend. However, RDF is highly heterogeneous, with its complex composition changing with season and source. This work is focused on the chemical characterization of six RDF samples listed as A, B, C, D, E, and F from different locations across the country using pyrolysis gas chromatography-mass spectrometry (Py-GC/MS), thermogravimetric instrument and CHONS elemental analyzer. Qualitative and semi-quantitative results were obtained using EGA, single-shot and double-shot analysis methods. EGA has been used to derive qualitative results in correlation with TGA. FTIR analysis was carried out to understand the initial functional characterization. XRF analysis along with ICP-MS triple quad revealed the inorganic makeup of the samples. Single-shot results indicated that apart from non-hydrocarbon gases (CO2 and O2), the long-chain alkenes were most abundant, followed by alkanes, aromatic compounds, and ketones. Double-shot analysis indicated the presence of chloride and sulphur-based compounds in RDF samples as limiting components. Polystyrene contribution to LHV of RDF has been highlighted. The study can be further used to conduct compositional studies of RDF and its pyrolytic behavior in the gasifier. It will further strengthen the future gasifier models where pyrolysis is one of the critical steps. This technique will be very helpful in obtaining a better characterization of volatile and non-volatile (bio-oil and ash) fractions of the pyrolytic products.

Identifying promoter sequence architectures via a chunking-based algorithm using non-negative matrix factorisation.

Core promoters are stretches of DNA at the beginning of genes that contain information that facilitates the binding of transcription initiation complexes. Different functional subsets of genes have core promoters with distinct architectures and characteristic motifs. Some of these motifs inform the selection of transcription start sites (TSS). By discovering motifs with fixed distances from known TSS positions, we could in principle classify promoters into different functional groups. Due to the variability and overlap of architectures, promoter classification is a difficult task that requires new approaches. In this study, we present a new method based on non-negative matrix factorisation (NMF) and the associated software called seqArchR that clusters promoter sequences based on their motifs at near-fixed distances from a reference point, such as TSS. When combined with experimental data from CAGE, seqArchR can efficiently identify TSS-directing motifs, including known ones like TATA, DPE, and nucleosome positioning signal, as well as novel lineage-specific motifs and the function of genes associated with them. By using seqArchR on developmental time courses, we reveal how relative use of promoter architectures changes over time with stage-specific expression. seqArchR is a powerful tool for initial genome-wide classification and functional characterisation of promoters. Its use cases are more general: it can also be used to discover any motifs at near-fixed distances from a reference point, even if they are present in only a small subset of sequences.

Characterization of a novel aromatic substrate-processing microcompartment in Actinobacteria.

We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta-cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase's ability to cleave both 2-aminophenol and catechol in vitro, in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta-cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.

Disease Impact and Burden in Patients with SCN2A-Related Developmental and Epileptic Encephalopathy (P11-9.012)

<h3>Objective:</h3> Explore the burden of <i>SCN2A</i>-DEE on patients and caregivers using clinical data generated via a novel, patient-consented, real-world data platform. <h3>Background:</h3> <i>SCN2A</i>-related DEE comprises a subset of rare neurodevelopmental genetic disorders characterized by significant heterogeneity and limited understanding of disease burden and progression. <h3>Design/Methods:</h3> Real-world clinical data were extracted from patients with an <i>SCN2A</i> variant using Invitae’s Ciitizen platform, which employs a proprietary approach combining machine learning and expert human review to streamline generation of research-grade data from unstructured health records spanning ~10 years. Extracted data (seizure history, medications, comorbidities, developmental milestones) were classified by phenotype based on age at seizure onset, type of seizure at initial presentation, variant type and functional characterization using dynamic action potential clamp analysis. <h3>Results:</h3> 45 patients (92%) had data available for analyses. Four phenotypes were identified across three groups: early onset (EO, 33.5%), late onset (LO, 42%), and autism without epilepsy (AO, 24.5%). The LO group contained two distinct phenotypes based on age and type of seizure at onset: the first, characterized by initial seizure presentation with infantile spasms (LOIS, 20%); the second characterized by seizures other than infantile spasms and presenting later in life (LO, 22%). Mean patient age across cohorts was 8 years (0.8–23.3), with similar percentages of males and females. Mean age at seizure onset was 5.1 days (1–44) in the EO group. 53% of patients with seizures experienced status epilepticus at least once. Mean number of medications prescribed over a patient’s lifetime was 18.1 (0–58). Patients had multiple comorbidities, with global developmental delay the most common (97%), and hypotonia, GERD, autism, and sleep disorders reported in &gt;50%. All patients did not meet at least one critical developmental milestone. <h3>Conclusions:</h3> Using a novel real-world data platform and functional variant characterization, we provide unprecedented insight into clinical phenotypes, disease burden and treatment patterns in <i>SCN2A</i>-DEE. <b>Disclosure:</b> Kelley Dalby has received personal compensation for serving as an employee of Praxis Precision Medicines. Kelley Dalby has stock in Praxis Precision Medicines. Kelley Dalby has stock in RogCon. Mr. Snyder has received personal compensation for serving as an employee of Praxis Precision Medicines. Mr. Snyder has received stock or an ownership interest from Praxis Precision Medicines. Dr. G Matthews has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Praxis Precision Medicines. Dr. G Matthews has stock in Praxis Precision Medicines. Ms. Brimble has received personal compensation for serving as an employee of Invitae. Ms. Brimble has stock in Invitae. Ms. Brimble has stock in Ciitizen. Mr. Beek has received personal compensation for serving as an employee of invitae. Mr. Beek has stock in invitae. Ms. Berk has received personal compensation for serving as an employee of Invitae. Ms. Berk has stock in Invitae. Dr. Oldham has received personal compensation for serving as an employee of Praxis Precision Medicines. Dr. Souza has received personal compensation for serving as an employee of Praxis Precision Medicines. Dr. Souza has stock in Praxis Precision Medicines. Steven Petrou has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Praxis Precision Medicines. Steven Petrou has stock in Praxis Precision Medicines. The institution of Steven Petrou has received research support from Praxis Precision Medicines. The institution of Steven Petrou has received research support from Medical Research Future Fund. Steven Petrou has received intellectual property interests from a discovery or technology relating to health care. Steven Petrou has received intellectual property interests from a discovery or technology relating to health care.

Identification of candidate genes in cotton associated with specific seed traits and their initial functional characterization in Arabidopsis

Oilseed crops are used to produce vegetable oil to satisfy the requirements of humans and livestock. Cotton (Gossypium spp.) is of great economic value because it is used as both an important textile commodity and a nutrient-rich resource. Cottonseed oil is rich in polyunsaturated fatty acids and does not contain trans fatty acids; hence, it is considered a healthy vegetable oil. However, research on the genetic basis for cottonseed protein content, oil production, and fatty acid composition is lacking. Here, we investigated the protein content, oil content, and fatty acid composition in terms of oleic acid (C18:1) and linoleic acid (C18:2) in mature cottonseeds from 318 Gossypium hirsutum accessions. Moreover, we examined the dynamic change of protein content and lipid composition including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) in developing seeds from 258 accessions at 10 and 20 days post-anthesis. Then, we conducted a genome-wide association study and identified 152 trait-associated loci and 64 candidate genes responsible for protein and oil-related contents in mature cottonseeds and ovules. Finally, six candidate genes were experimentally validated to be involved in the regulation of fatty acid biosynthesis through heterologous expression in Arabidopsis. These results comprise a solid foundation for expanding our understanding of lipid biosynthesis in cotton, which will help breeders manipulate protein and oil contents to make it a fully developed 'fiber, food, and oil crop'.

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Expression of the Malus sieversii NF-YB21 Encoded Gene Confers Tolerance to Osmotic Stresses in Arabidopsis thaliana.

Drought is the main environmental factor that limits the yield and quality of apples (Malus × domestica) grown in arid and semi-arid regions. Nuclear factor Ys (NF-Ys) are important transcription factors involved in the regulation of plant growth, development, and various stress responses. However, the function of NF-Y genes is poorly understood in apples. Here, we identified 43 NF-Y genes in the genome of apples and conducted an initial functional characterization of the apple NF-Y. Expression analysis of NF-Y members in M. sieversii revealed that a large number of NF-Ys were highly expressed in the roots compared with the leaves, and a large proportion of NF-Y genes responded to drought treatment. Furthermore, heterologous expression of MsNF-YB21, which was significantly upregulated by drought, led to a longer root length and, thus, conferred improved osmotic and salt tolerance in Arabidopsis. Moreover, the physiological analysis of MsNF-YB21 overexpression revealed enhanced antioxidant systems, including antioxidant enzymes and compatible solutes. In addition, genes encoding catalase (AtCAT2, AtCAT3), superoxide dismutase (AtFSD1, AtFSD3, AtCSD1), and peroxidase (AtPER12, AtPER42, AtPER47, AtPER51) showed upregulated expression in the MsNF-YB21 overexpression lines. These results for the MsNF-Y gene family provide useful information for future studies on NF-Ys in apples, and the functional analysis of MsNF-YB21 supports it as a potential target in the improvement of apple drought tolerance via biotechnological strategies.

Long Noncoding RNA Cardiac Physiological Hypertrophy-Associated Regulator Induces Cardiac Physiological Hypertrophy and Promotes Functional Recovery After Myocardial Ischemia-Reperfusion Injury.

The benefits of exercise training in the cardiovascular system have been well accepted; however, the underlying mechanism remains to be explored. Here, we report the initial functional characterization of an exercise-induced cardiac physiological hypertrophy-associated novel long noncoding RNA (lncRNA). Using lncRNA microarray profiling, we identified lncRNAs in contributing the modulation of exercise-induced cardiac growth that we termed cardiac physiological hypertrophy-associated regulator (CPhar). Mice with adeno-associated virus serotype 9 driving CPhar overexpression and knockdown were used in in vivo experiments. Swim training was used to induce physiological cardiac hypertrophy in mice, and ischemia reperfusion injury surgery was conducted to investigate the protective effects of CPhar in mice. To investigate the mechanisms of CPhar's function, we performed various analyses including quantitative reverse transcription polymerase chain reaction, Western blot, histology, cardiac function (by echocardiography), functional rescue experiments, mass spectrometry, in vitro RNA transcription, RNA pulldown, RNA immunoprecipitation, chromatin immunoprecipitation assay, luciferase reporter assay, and coimmunoprecipitation assays. We screened the lncRNAs in contributing the modulation of exercise-induced cardiac growth through lncRNA microarray profiling and found that CPhar was increased with exercise and was necessary for exercise-induced physiological cardiac growth. The gain and loss of function of CPhar regulated the expression of proliferation markers, hypertrophy, and apoptosis in cultured neonatal mouse cardiomyocytes. Overexpression of CPhar prevented myocardial ischemia reperfusion injury and cardiac dysfunction in vivo. We identified DDX17 (DEAD-Box Helicase 17) as a binding partner of CPhar in regulating CPhar downstream factor ATF7 (activating transcription factor 7) by sequestering C/EBPβ (CCAAT/enhancer binding protein beta). Our study of this lncRNA CPhar provides new insights into the regulation of exercise-induced cardiac physiological growth, demonstrating the cardioprotective role of CPhar in the heart, and expanding our mechanistic understanding of lncRNA function, as well.

SOGA1 and SOGA2/MTCL1 are CLASP-interacting proteins required for faithful chromosome segregation in human cells.

CLASPs are key modulators of microtubule dynamics throughout the cell cycle. During mitosis, CLASPs independently associate with growing microtubule plus-ends and kinetochores and play essential roles in chromosome segregation. In a proteomic survey for human CLASP1-interacting proteins during mitosis, we have previously identified SOGA1 and SOGA2/MTCL1, whose mitotic roles remained uncharacterized. Here we performed an initial functional characterization of human SOGA1 and SOGA2/MTCL1 during mitosis. Using specific polyclonal antibodies raised against SOGA proteins, we confirmed their expression and reciprocal interaction with CLASP1 and CLASP2 during mitosis. In addition, we found that both SOGA1 and SOGA2/MTCL1 are phospho-regulated during mitosis by CDK1. Immunofluorescence analysis revealed that SOGA2/MTCL1 co-localizes with mitotic spindle microtubules and spindle poles throughout mitosis and both SOGA proteins are enriched at the midbody during mitotic exit/cytokinesis. GFP-tagging of SOGA2/MTCL1 further revealed a microtubule-independent localization at kinetochores. Live-cell imaging after siRNA-mediated knockdown of SOGA1 and SOGA2/MTCL1 showed that they are independently required for distinct aspects of chromosome segregation. Thus, SOGA1 and SOGA2/MTCL1 are bona fide CLASP-interacting proteins during mitosis required for faithful chromosome segregation in human cells.

Translating genetic risk variants in disease‐associated enhancers into novel mouse models of Alzheimer’s disease

AbstractBackgroundThe enrichment of late‐onset Alzheimer’s disease (LOAD) GWAS variants in noncoding regions of the genome reveals new potential for modeling disease risk. Yet, identifying noncoding causal variants and the cell types in which they are functional remains challenging. Translating noncoding variants into novel mouse models can elucidate phenotypic effects of those variants through specific perturbations of gene enhancers associated with LOAD risk.MethodWe developed a multi‐tier approach to translate noncoding GWAS risk variants into novel mouse models of LOAD. First, we used publicly available GWAS summary statistics and eQTL data from the AMP‐AD consortium (ROS/MAP, Mayo Clinic) across two brain regions (DLPFC, TCX) to prioritize LOAD risk variants. Then, a deep learning approach (DeepSEA) was implemented to identify regulatory variants in conserved enhancers that are likely to disrupt expression of nearby candidate genes in both human and mouse. Further, ATAC‐seq data from human and mouse was integrated to predict cell‐type specific effects of the prioritized regulatory elements. Finally, the top‐predicted disease‐associated variants were used to develop novel mouse models as part of the MODEL‐AD initiative.ResultOur integrative approach for prioritizing disease‐associated variants revealed a set of conserved risk variants that mediate the expression of nearby LOAD candidate genes in a cell type‐specific manner. These included several well‐known LOAD risk loci (BIN1, PT2KB, SCIMP, CD2AP). In addition, we identified a genome‐wide significant risk variant (rs4575098, p = 2.05 × 10(‐11), Jansen et al. 2019), which decreased expression of the novel candidate FCER1G in the brain of LOAD patients (ROS/MAP eQTL p = 1.81 × 10(‐05), beta = ‐0.27). This highly conserved variant resides in a microglia specific enhancer and overlaps with a binding site of the transcriptional regulator PU.1 (SPI1). We created a 2kb enhancer deletion mouse model for the FCER1G associated variant and knock‐in models for variants linked to increased expression in three well‐known GWAS loci (BIN1, SCIMP, PT2KB). We provide initial functional characterization for our novel models based on RNA‐seq data.ConclusionOur findings expand the list of LOAD candidate genes likely to be impacted by noncoding variants and provide a novel set of mouse models to study their effects in vivo.

Application of target repositioning and in silico screening to exploit fatty acid binding proteins (FABPs) from Echinococcus multilocularis as possible drug targets.

Fatty acid binding proteins (FABPs) are small intracellular proteins that reversibly bind fatty acids and other hydrophobic ligands. In cestodes, due to their inability to synthesise fatty acids and cholesterol de novo, FABPs, together with other lipid binding proteins, have been proposed as essential, involved in the trafficking and delivery of such lipophilic metabolites. Pharmacological agents that modify specific parasite FABP function may provide control of lipid signalling pathways, inflammatory responses and metabolic regulation that could be of crucial importance for the parasite development and survival. Echinococcus multilocularis and Echinococcus granulosus are, respectively, the causative agents of alveolar and cystic echinococcosis (or hydatidosis). These diseases are included in the World Health Organization's list of priority neglected tropical diseases. Here, we explore the potential of FABPs from cestodes as drug targets. To this end, we have applied a target repurposing approach to identify novel inhibitors of Echinococcus spp. FABPs. An ensemble of computational models was developed and applied in a virtual screening campaign of DrugBank library. 21 hits belonging to the applicability domain of the ensemble models were identified, and 3 of the hits were assayed against purified E. multilocularis FABP, experimentally confirming the model's predictions. Noteworthy, this is to our best knowledge the first report on isolation and purification of such four FABP, for which initial structural and functional characterization is reported here.

Transcriptional control of a novel long noncoding RNA Mymsl in smooth muscle cells by a single Cis-element and its initial functional characterization in vessels

Differentiated vascular smooth muscle cells (VSMCs) are crucial in maintaining vascular homeostasis. While the coding transcriptome of the differentiated VSMC phenotype has been defined, we know little about its noncoding signature. Herein, we identified a Myocardin-induced muscle specific long noncoding RNA (lncRNA) (Mymsl) downregulated upon VSMC phenotypic modulation. We demonstrated an essential role of a proximal consensus CArG element in response to MYOCD/SRF in vitro. To validate the in vivo role of this CArG element, we generated CArG mutant mice via CRISPR-Cas9 genome editing. While the CArG mutation had no impact on the expression of surrounding genes, it abolished Mymsl expression in SMCs, but not skeletal and cardiac muscle. Chromatin immunoprecipitation assays (ChIPs) showed decreased SRF binding to CArG region in mutants whereas the enrichment of H3K79Me2 remained the same. RNA-seq analysis showed a downregulation of matrix genes in aortas from Mymsl knockout mice, which was further validated in injured carotid arteries. Our study defined the transcriptional control of a novel lncRNA in SMCs via a single transcription factor binding site, which may offer a new strategy for generating SMC-specific knockout mouse models. We also provided in vivo evidence supporting the potential importance of Mymsl in vascular pathophysiology.

Controlled Reduction of Genomic Heterozygosity in an Industrial Yeast Strain Reveals Wide Cryptic Phenotypic Variation.

Abundant genomic heterozygosity can be found in wild strains of the budding yeast Saccharomyces cerevisiae isolated from industrial and clinical environments. The extent to which heterozygosity influences the phenotypes of these isolates is not fully understood. One such case is the PE-2/JAY270 strain, a natural hybrid widely adopted by sugarcane bioethanol distilleries for its ability to thrive under harsh biotic and abiotic stresses during industrial scale fermentation, however, it is not known whether or how the heterozygous configuration of the JAY270 genome contributes to its many desirable traits. In this study, we took a step toward exploring this question by conducting an initial functional characterization of JAY270’s heteroalleles. We manipulated the abundance and distribution of heterozygous alleles through inbreeding and targeted uniparental disomy (UPD). Unique combinations of homozygous alleles in each inbred strain revealed wide phenotypic variation for at least two important industrial traits: Heat stress tolerance and competitive growth. Quantitative trait loci analyses allowed the identification of broad genomic regions where genetic polymorphisms potentially impacted these traits, and there was no overlap between the loci associated with each. In addition, we adapted an approach to induce bidirectional UPD of three targeted pairs of chromosomes (IV, XIV, and XV), while heterozygosity was maintained elsewhere in the genome. In most cases UPD led to detectable phenotypic alterations, often in opposite directions between the two homozygous haplotypes in each UPD pair. Our results showed that both widespread and regional homozygosity could uncover cryptic phenotypic variation supported by the heteroalleles residing in the JAY270 genome. Interestingly, we characterized multiple examples of inbred and UPD strains that displayed heat tolerance or competitive growth phenotypes that were superior to their heterozygous parent. However, we propose that homozygosity for those regions may be associated with a decrease in overall fitness in the complex and dynamic distillery environment, and that may have contributed to slowing down the erosion of heterozygosity from the JAY270 genome. This study also laid a foundation for approaches that can be expanded to the identification of specific alleles of interest for industrial applications in this and other hybrid yeast strains.

Characterization of Maize Phytochrome-Interacting Factors in Light Signaling and Photomorphogenesis.

Increasing planting density has been an effective means of increasing maize (Zea mays ssp. mays) yield per unit of land area over the past few decades. However, high-density planting will cause a reduction in the ratio of red to far-red incident light, which could trigger the shade avoidance syndrome and reduce yield. The molecular mechanisms regulating the shade avoidance syndrome are well established in Arabidopsis (Arabidopsis thaliana) but poorly understood in maize. Here, we conducted an initial functional characterization of the maize Phytochrome-Interacting Factor (PIF) gene family in regulating light signaling and photomorphogenesis. The maize genome contains seven distinct PIF genes, which could be grouped into three subfamilies: ZmPIF3s, ZmPIF4s, and ZmPIF5s Similar to the Arabidopsis PIFs, all ZmPIF proteins are exclusively localized to the nucleus and most of them can form nuclear bodies upon light irradiation. We show that all of the ZmPIF proteins could interact with ZmphyB. Heterologous expression of each ZmPIF member could partially or fully rescue the phenotype of the Arabidopsis pifq mutant, and some of these proteins conferred enhanced shade avoidance syndrome in Arabidopsis. Interestingly, all ZmPIF proteins expressed in Arabidopsis are much more stable than their Arabidopsis counterparts upon exposure to red light. Moreover, the Zmpif3, Zmpif4, and Zmpif5 knockout mutants generated via CRISPR/Cas9 technology all showed severely suppressed mesocotyl elongation in dark-grown seedlings and were less responsive to simulated shade treatment. Taken together, our results reveal both conserved and distinct molecular properties of ZmPIFs in regulating light signaling and photomorphogenesis in maize.

Abstract 909: Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9

Abstract Background: Androgen receptor (AR) is the key drug target in prostate cancer. AR splice variant-7 (AR-V7) is the most relevant AR variants implicated in castration-resistant prostate cancer (CRPC). Detection of AR-V7 in both liquid biopsies and tissue biopsies predicts poorer response to novel AR-targeting drugs. However, because AR-V7 always coexists with the full-length AR (AR-FL), the functional importance of AR-V7 needs to be dissected in the context of AR-FL. Lack of relevant cell line models presented a hurdle for detailed dissection of the role for AR-V7. It is essential to develop cell lines that express endogenous AR-V7 at a level comparable to clinical specimens, with or without AR-FL, to facilitate functional studies. The goal of this study is to develop and characterize LNCaP95 lines following specific and complete knock-out of AR molecules by CRISPR-Cas9 system. Methods: Different AR isoforms were knocked-out in parental LNCaP95 cells by CRISPR-Cas9. AR-FL was knocked-out through sgRNAs targeting AR exon5 or exon6. AR isoforms with functional N-terminal domain (total AR) were knocked-out through sgRNA targeting AR exon1. Complete knockout of AR-FL and total AR was achieved in LNCaP95 cells. The on-target editing of AR genomic regions were verified by Sanger Sequencing. Results: Complete knockout of AR-FL protein and total AR protein was confirmed in two clones carrying homologous indels, ARfl-KO#1/2 and AR-KO#1/2. Antibodies against different AR domains were used to differentiate expression of different AR isoforms, by Western blot and immunohistochemistry (IHC). Cell proliferation was not affected by androgen in AR-FL or total AR knockout cells, as shown in cell growth and clonogenic assays. The expression of AR-target genes such as prostate-specific antigen (PSA) were analyzed by realtime-PCR. PSA was expressed at similar level compared to parental cells in AR-FL knockout cells, which was totally lost in total AR knockout cells. Interestingly, total AR knockout cells are more resistant to ionizing radiation and PARP inhibitor olaparib compared to parental cell line. Conclusion: We have successfully developed the first prostate cancer cell line expressing endogenous AR-V7 at a level comparable to clinical specimens without AR-FL, allowing dissection of the functions mediated by AR-FL and AR-V7. Initial functional characterization suggested AR-V7 maintained the baseline level of AR signaling activity, which was not dependent on androgen. The chromatin-binding pattern of AR-V7 in the absence of AR-FL is under investigation. We have also developed a novel total AR knockout cell model from the AR-positive parental cell line. This model suggested prostate cancer cells were able to survive in an AR-null manner, although loss of AR significantly inhibited cell proliferation. Preliminary studies have found loss of total AR confers resistance to ionizing radiation and PARP inhibition. Citation Format: Yezi Zhu, Pei Zhao, Changxue Lu, Shihua Chen, Jun Luo. Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 909.

Indole Biodegradation in Acinetobacter sp. Strain O153: Genetic and Biochemical Characterization

Indole is a molecule of considerable biochemical significance, acting as both an interspecies signal molecule and a building block of biological elements. Bacterial indole degradation has been demonstrated for a number of cases; however, very little is known about genes and proteins involved in this process. This study reports the cloning and initial functional characterization of genes (iif and ant cluster) responsible for indole biodegradation in Acinetobacter sp. strain O153. The catabolic cascade was reconstituted in vitro with recombinant proteins, and each protein was assigned an enzymatic function. Degradation starts with oxidation, mediated by the IifC and IifD flavin-dependent two-component oxygenase system. Formation of indigo is prevented by IifB, and the final product, anthranilic acid, is formed by IifA, an enzyme which is both structurally and functionally comparable to cofactor-independent oxygenases. Moreover, the iif cluster was identified in the genomes of a wide range of bacteria, suggesting the potential of widespread Iif-mediated indole degradation. This work provides novel insights into the genetic background of microbial indole biodegradation.IMPORTANCE The key finding of this research is identification of the genes responsible for microbial biodegradation of indole, a toxic N-heterocyclic compound. A large amount of indole is present in urban wastewater and sewage sludge, creating a demand for an efficient and eco-friendly means to eliminate this pollutant. A common strategy of oxidizing indole to indigo has the major drawback of producing insoluble material. Genes and proteins of Acinetobacter sp. strain O153 (DSM 103907) reported here pave the way for effective and indigo-free indole removal. In addition, this work suggests possible novel means of indole-mediated bacterial interactions and provides the basis for future research on indole metabolism.

Abstract 343: Proteomic and functional studies identify DCAF7 as major partner of DYRK1A

Abstract Human DYRK1A protein kinase is encoded by dosage-dependent gene since an extra copy contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe developmental defects. Current knowledge of DYRK1A’s role in phosphorylation of proteins involved in cell cycle control, transcription and tumor suppression does not fully explain dosage-dependent function of this important kinase. Using MudPIT proteomic analysis, we identified DYRK1A interacting proteins in human T98G cell line. Four independent DYRK1A-HA pull-down samples were analyzed to identify 50 proteins that were specifically detected in 3 out of 4 replicates. This analysis identified WD-repeat protein DCAF7 as major partner of DYRK1A that was most highly enriched among DYRK1A-binding proteins. Furthermore, glycerol gradient ultracentrifugation showed almost exact co-fractionation of DCAF7 and DYRK1A. DCAF7 has been shown to bind several protein kinases including DYRK1A, DYRK1B, HIPK2 and MAP3K1 suggesting that it serves as a scaffold protein in signal transduction. However, the function of DCAF7 is not established yet. In order to understand the functional relationship between DCAF7 and DYRK1A, we undertook MudPIT proteomic analysis of DCAF7 binding proteins in T98G cells and compared the data with the DYRK1A interactome dataset. Analysis of three independent DCAF7-HA pull-down samples identified 32 proteins specifically detected in all three replicates including DCAF7, DYRK1A and five proteins earlier detected in DYRK1A MudPIT analysis. Given the proposed role of DCAF7 as a scaffold protein, we tested whether DCAF7 mediates its interaction with some of its interacting proteins. Interestingly, we found that RNF169, a recently characterized RING-domain ubiquitin ligase involved in the DNA double-strand break (DSB) repair, binds to N-terminus of DYRK1A independently of DCAF7. Furthermore, our data show that DYRK1A is required for DCAF7-RNF169 interaction because this interaction was abolished in human U-2 OS cells where DYRK1A gene was disrupted using CRISPR-Cas9 editing (DYRK1A-KO cells). Interestingly, stable overexpression of DCAF7 in DYRK1A-KO U-2 OS cells rescued some of the phenotypes observed in these cells, suggesting that DCAF7 could be an effector downstream of DYRK1A. Our findings revealed an unexpected scaffolding role of DYRK1A that is required for mediating the interaction between DNA repair protein RNF169 and the WD-repeat protein DCAF7. While further studies are needed to understand the exact role of the RNF169-DCAF7 interaction in DNA repair, the novel role of DYRK1A as a scaffold protein could explain some aspects of the dosage-dependent function of this protein kinase. In addition, we report here an initial functional characterization of the DCAF7-DYRK1A interaction in human cells and discuss novel functional interactions of DCAF7 that could be independent of DYRK1A. Citation Format: Varsha Anathapadmanabhan, Selene Swanson, Siddharth Saini, Vijay Menon, Larisa Litovchick. Proteomic and functional studies identify DCAF7 as major partner of DYRK1A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2017-343