Higher Organisms Research Articles

Targeting APC/C Ubiquitin E3-Ligase Activation with Pyrimidinethylcarbamate Apcin Analogues for the Treatment of Breast Cancer

Activation of the ubiquitin ligase APC/C by the protein Cdc20 is an essential requirement for proper cell division in higher organisms, including humans. APC/C is the ultimate effector of the Spindle Assembly Checkpoint (SAC), the signalling system that monitors the proper attachment of chromosomes to microtubules during cell division. Defects in this process result in genome instability and cancer. Interfering with APC/C substrate ubiquitylation in cancer cells delays mitotic exit, which induces cell death. Therefore, impairing APC/C function represents an opportunity for the treatment of cancer and malignancies associated with SAC dysregulation. In this study, we report a new class of pyrimidinethylcarbamate apcin analogues that interfere with APC/C activity in 2D and 3D breast cancer cells. The new pyrimidinethylcarbamate apcin analogues exhibited higher cytotoxicity than apcin in all breast cancer cell subtypes investigated, with much lower cytotoxicity observed in fibroblasts and RPE-1 cells. Further molecular rationalisation of apcin and its derivatives was conducted using molecular docking studies. These structural modifications selected from the in silico studies provide a rational basis for the development of more potent chemotypes to treat highly aggressive breast cancer and possibly other aggressive tumour types of diverse tissue origins.

The biochemical effects of carotenoids in orange carrots on the colonic proteome in a mouse model of diet-induced obesity

IntroductionCarotenoids are naturally occurring pigments in plants and are responsible for the orange, yellow, and red color of fruits and vegetables. Carrots are one of the primary dietary sources of carotenoids. The biological activities of carotenoids in higher organisms, including their immunomodulatory activities, are well documented in most tissues but not the large intestine. The gastrointestinal barrier acts as a line of defense against the systemic invasion of pathogenic bacteria, especially at the colonic level.MethodsTo test whether carotenoids in orange carrots can alleviate obesity-associated gut inflammation and strengthen the intestinal barrier function, male C57BL/6J mice were randomized to one of four experimental diets for 20 weeks (n = 20 animals/group): Low-fat diet (LFD, 10% calories from fat), high-fat diet (HFD, 45% calories from fat), HFD with white carrot powder (HFD+WC), or HFD with orange carrot powder (HFD + OC). Colon tissues were harvested to analyze the biochemical effects of carotenoids in carrots. The distal sections were subjected to isobaric labeling-based quantitative proteomics in which tryptic peptides were labeled with tandem mass tags, followed by fractionation and LC-MS/MS analysis in an Orbitrap Eclipse Tribrid instrument.ResultsHigh-performance liquid chromatography results revealed that the HFD+WC pellets were carotenoid-deficient, and the HFD+OC pellets contained high concentrations of provitamin A carotenoids, specifically α-carotene and β-carotene. As a result of the quantitative proteomics, a total of 4410 differentially expressed proteins were identified. Intestinal barrier-associated proteins were highly upregulated in the HFD+OC group, particularly mucin-2 (MUC-2). Upon closer investigation into mucosal activity, other proteins related to MUC-2 functionality and tight junction management were upregulated by the HFD+OC dietary intervention.DiscussionCollectively, our findings suggest that carotenoid-rich foods can prevent high-fat diet-induced intestinal barrier disruption by promoting colonic mucus synthesis and secretion in mammalian organisms. Data are available via ProteomeXchange with identifier PXD054150.

Directionality theory and the origin of life.

The origin of cellular life can be described in terms of the transition from inorganic matter to the emergence of cooperative assemblies of organic matter: DNA and proteins, capable of replication and metabolism. Directionality theory is a mathematical theory of the collective behaviour of networks of organic matter: activated macromolecules, cells and higher organisms. Evolutionary entropy, a generalization of the thermodynamic entropy of Boltzmann, is a statistical measure of the cooperativity of the biotic components. The cornerstone of Directionality theory is the Entropic Principle of Evolution: evolutionary entropy increases in systems driven by a stable energy source, and decreases in systems subject to a fluctuating energy source. This article invokes the Entropic Principle of Evolution-an extension to biological systems of the Second Law of Thermodynamics-to provide an adaptive rationale for the following sequence of transformations that define the emergence of cellular life: (i) the self-assembly of activated macromolecules from inorganic matter; (ii) the emergence of an RNA world, defined by RNA molecules with catalytic and replicative properties; and (iii) the origin of cellular life, the integration of the three carbon-based polymers-DNA, proteins and lipids, to generate a metabolic and replicative unit.

Tail Tales: What We Have Learned About Regeneration from Xenopus Laevis Tadpoles.

This review explores the regenerative capacity of Xenopus laevis, focusing on tail regeneration, as a model to uncover cellular, molecular, and developmental mechanisms underlying tissue repair. X. laevis tadpoles provide unique insights into regenerative biology due to their regeneration-competent and -incompetent stages and ability to regrow complex structures in the tail, including the spinal cord, muscle, and skin, after amputation. The review delves into the roles of key signaling pathways, such as those involving reactive oxygen species (ROS) and signaling molecules like BMPs and FGFs, in orchestrating cellular responses during regeneration. It also examines how mechanotransduction, epigenetic regulation, and metabolic shifts influence tissue restoration. Comparisons of regenerative capacity with other species shed light on the evolutionary loss of regenerative abilities and underscore X. laevis as an invaluable model for understanding the constraints of tissue repair in higher organisms. This comprehensive review synthesizes recent findings, suggesting future directions for exploring regeneration mechanisms, with potential implications for advancing regenerative medicine.

Activation of the aryl hydrocarbon receptor via indole derivatives is a common feature in skin bacterial isolates.

The aryl hydrocarbon receptor (AhR) is a ligand-activated receptor implicated in many inflammatory disorders. The skin microbiota plays a crucial role in maintaining epidermal barrier integrity and is thought to modulate skin homeostasis partly through the production of AhR ligands, including metabolites of microbial tryptophan metabolism such as indole derivatives. Here, we report the skin microbiota that activate AhR and their unique tryptophan metabolite profiles. Of the bacteria isolated from healthy human skin and screened for the ability to metabolize tryptophan (18 species, five genera), 14 were positive. The tryptophan metabolites of 10 positive and two negative bacteria were then characterized using liquid chromatography-mass spectrometry. Whole genome sequencing confirmed the presence of key genes involved in the indole-3-pyruvic acid pathway within the genomes of indole-3-acetaldehyde, indole-3-acetic acid, and indole-3-aldehyde-producing organisms. A cell-based luciferase reporter gene assay identified functional agonist activity against human AhR in the culture supernatants of 12 of the 18 species tested. High indole derivative-producing organisms induced potent AhR activation. These data demonstrate the relationship between skin microbiota, tryptophan metabolites, and AhR activation.

Convergent evolution links molybdenum insertase domains with organism-specific sequences

In all domains of life, the biosynthesis of the pterin-based Molybdenum cofactor (Moco) is crucial. Molybdenum (Mo) becomes biologically active by integrating into a unique pyranopterin scaffold, forming Moco. The final two steps of Moco biosynthesis are catalyzed by the two-domain enzyme Mo insertase, linked by gene fusion in higher organisms. Despite well-understood Moco biosynthesis, the evolutionary significance of Mo insertase fusion remains unclear. Here, we present findings from Neurospora crassa that shed light on the critical role of Mo insertase fusion in eukaryotes. Substituting the linkage region with sequences from other species resulted in Moco deficiency, and separate expression of domains, as seen in lower organisms, failed to rescue deficient strains. Stepwise truncation and structural modeling revealed a crucial 20-amino acid sequence within the linkage region essential for fungal growth. Our findings highlight the evolutionary importance of gene fusion and specific sequence composition in eukaryotic Mo insertases.

Roles of the Dbl family of RhoGEFs in mechanotransduction - a review.

Rho guanine nucleotide exchange factors (RhoGEFs) comprise a wide range of proteins with a common domain responsible for the activation of the Rho family of small GTPases and various domains in other regions. The evolutionary divergence of RhoGEFs enables actin cytoskeletal reorganization, leading to complex cellular responses in higher organisms. In this review, we address the involvement of RhoGEFs in the mechanical stress response of mammalian cells. The cellular mechanical stress response is essential for the proper and orderly regulation of cell populations, including the maintenance of homeostasis, tissue morphogenesis, and adaptation to the mechanical environment. In particular, this review focuses on the recent findings regarding the Dbl family of RhoGEFs involved in mechanical stress responses at the cell-cell and cell-substrate adhesion sites, and their molecular mechanisms underlying actin cytoskeleton remodeling and signal transduction.

On the chemistry of sunlight-induced DNA lesions: A perspective on the alkaline chemical-induced reactivities of photo-damaged pyrimidine intra-strand dimers.

Photoexcitation of cellular as well as isolated DNAs upon exposure to the UV portion of sunlight or other UV sources can lead to the covalent dimerization of adjacent intra-strand stacked pyrimidine nucleobase rings (i.e., at 5'-Py-p-Py-3' sites). These modifications generate, in mammalian DNA as well as the DNA of all other forms of life, lesions such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs); and, in bacterial endospores, spore photoproducts (SPs). Importantly, the lesions formed in higher organisms can lead to disease states including cancer. While the formation, structure, and biological outcomes of pyrimidine dimer lesions have been the focus of much research, less has been known about their fundamental chemical properties until recently. Such an understanding of these lesions may lead to novel means to chemically identify and quantitate their presence in the genome. This review is intended to provide an overview of intra-strand pyrimidine dimer lesions derived from 5'-T-p-T sites with a focus on presenting what is currently known about their individual invitro alkaline chemical reactivities. Included here are descriptions of investigations of the DNA lesions CPD, 6-4PP, and SP, and, for comparison, the monomeric pyrimidine lesion 5,6-dihydo-2'-deoxyuridine (dHdU). Of interest, the alkaline hydrolyses of these various lesions are all found to be centered on the loss of aromaticity of a lesion Py ring (T) leading to a carbonyl "hot spot," the focal point of initial hydrolytic attack.

Genomic and phenotypic polymorphism of Clostridium botulinum Group II strain Beluga through laboratory domestication

Laboratory domestication is the result of genetic and physiological changes of organisms acquired during numerous passages in vitro. This phenomenon has been observed in bacteria as well as in higher organisms. In an effort to understand the impact of laboratory domestication on the foodborne pathogen Clostridium botulinum and related microbial food safety research, we investigated multiple spore stocks of C. botulinum Group II Beluga from our collection, as that is a widely applied model strain used in laboratories over decades. An acquired nutrient auxotrophy was confirmed as thymidine dependency using phenotypic microarrays. In parallel, whole-genome re-sequencing of all stocks revealed a mutation in thyA encoding thymidylate synthase essential for de-novo synthesis of dTMP from dUMP in the auxotrophic stocks. A thyA-deficient Beluga variant stock was successfully complemented by introducing an intact variant of thyA and thymidine prototrophy was restored, indicating that the thymidine auxotrophy was solely due to the presence of a SNP in thyA. Our data suggested that this mutation, deleterious under nutrient-poor growth conditions in a chemically defined medium, has been present and maintained in laboratory stocks for nearly 30 years. Yet, the mutation remained unidentified since receiving the strain, most likely due to routine use of culture conditions optimized for growth performance. This work pinpoints the need for careful monitoring of model strains extensively used in laboratory settings at both phenotypic and genomic level. In applications like food safety challenge tests, compromised strains could cause incorrect predictions and thereby have deleterious consequences. To mitigate the risk of acquiring mutations, we recommend keeping passage numbers of laboratory strains low and to avoid single-colony passaging. In addition, relevant strains should be subjected to regular WGS checks and physiological validation to exclude DNA mutations with potential negative impacts on research data integrity and reproducibility.

Human anti-apoptotic Bcl-2 and Bcl-xL proteins protect yeast cells from aging induced oxidative stress

Aging is a degenerative, biological, and time-dependent process that affects all organisms. Yeast aging is a physiological phenomenon characterized by the progressive transformation of yeast cells, resulting in modifications to their viability and vitality. Aging in yeast cells is comparable to that in higher organisms in some respects; however, due to their straightforward and well-characterized genetic makeup, these cells present unique advantages when it comes to researching the aging process. Here, we assessed the impact of human anti-apoptotic Bcl-2 and Bcl-xL proteins on aging using a yeast model. The findings clearly showed that these proteins exhibited remarkable anti-aging properties in yeast cells. Our data indicate that the presence of both proteins enhanced the reproductive survival of aging cells, likely by effecting the components functioning as both pro- and anti-oxidants, depending on the stage of yeast cell lifespan. Both proteins partially protected yeast cells from aging-related morphological deformations and cellular damage during the aging period. In particular, Bcl-xL expressing yeast cells reached the maximum activity levels for almost all of the major antioxidant enzymes and the total antioxidant status on the 8th day of lifespan and could provide effective protection at the latest stage of the investigated aging period. The chemometric data analysis of IR spectra confirmed the findings of the morphological and biochemical analyses. In this regard, specifically, understanding the mechanism of action on the cellular redox state of Bcl-xL in yeast may facilitate comprehension of its indirect antioxidant function in higher eukaryotes.

Characterization of the Three DHFRs and K65P Variant: Enhanced Substrate Affinity and Molecular Dynamics Analysis.

Dihydrofolate reductase (DHFR) is ubiquitously present in all living organisms and plays a crucial role in the growth of the fungal pathogen R.solani. Sequence alignment confirmed the evolutionary conservation of the essential lid domain, with the amino acid 'P' within the PEKN lid domain appearing with a frequency of 89.5% in higher organisms and 11.8% in lower organisms. Consequently, a K65P variant was introduced into R.solani DHFR (rDHFR). Subsequent enzymatic kinetics assays were conducted for human DHFR (hDHFR), rDHFR, E. coli DHFR (eDHFR), and the K65P variant. hDHFR exhibited the highest kcat of 0.95s-1, followed by rDHFR with 0.14s-1, while eDHFR displayed the lowest kcat of 0.09s-1. Remarkably, the K65P variant induced a significant reduction in Km, resulting in a 1.8-fold enhancement in catalytic efficiency (kcat/Km) relative to the wild type. Differential scanning fluorimetry and binding free energy calculations confirmed the enhanced substrate affinity for both folate and NADPH in the K65P variant. These results suggest that the K65P mutation enhances substrate affinity and catalytic efficiency in DHFR, highlighting the evolutionary and functional importance of the K65 residue.

In situ imaging of a kleptoplastidic ciliate thin layer indicates traditional sampling underestimates oceanic mixotroph biomass

Planktonic mixotrophs, including ciliates, are abundant, yet understudied, mediators of primary and secondary production in aquatic food webs. Here, we report high-resolution in situ observations of a thin layer—a vertically limited patch of high organism abundance—of the ciliate, Laboea strobila. Within the thin layer, peak L. strobila abundances were as high as 62,000 cells/L, six times higher than previously reported, and were strongly positively correlated with co-measured chlorophyll a concentrations, indicating that the organism indulged in kleptoplasty, retaining plastids from ingested algae. Estimates indicated L. strobila contributed to ~63–78% of chlorophyll a within the layer, in sharp contrast to conventional expectations that phytoplankton dominate this signature, suggesting that mixotroph biomass, and consequently, their contribution to marine ecosystems is widely underestimated. These observations also highlight the importance of pairing traditional approaches such as chlorophyll a measurements with high-resolution imaging for proper attribution of biomass proxies.

Interplay of RNA m6A Modification-Related Geneset in Pan-Cancer.

Background: N6-methyladenosine (m6A), is the most common modification found in mRNA and lncRNA in higher organisms and plays an important role in physiology and pathology. However, its role in pan-cancer has not been explored. Results: A total of 31 m6A modification regulators, including 12 writers, 2 erasers, and 17 readers are identified in the current study. The functional analysis of the regulators results in the enrichment of processes, primarily related to RNA modification and metabolism, and the PPI network reveals multiple interactions among the regulators. The mRNA expression analysis reveals a high expression for most of the regulators in pan-cancer. Most of the m6A regulators are found to be mutated across the cancers, with ZC3H13, VIRMA, and PRRC2A having a higher frequency rate. Significant correlations of the regulators with clinicopathological parameters, such as age, gender, tumor stage, and grade are identified in pan-cancer. The m6A regulators' expression is found to have significant positive correlations with the miRNAs in pan-cancer. The expression pattern of the m6A regulators is able to classify the tumors into different subclusters as well as into high- and low-risk groups. These tumor groups show differential patterns in terms of their immune cell infiltration, tumor stemness score, genomic heterogeneity score, expression of immune regulatory/checkpoint genes, and correlations between the regulators and the drugs. Conclusions: Our study provide a comprehensive overview of the functional roles, genetic and epigenetic alterations, and prognostic value of the RNA m6A regulators in pan-cancer.

A TBK1-independent primordial function of STING in lysosomal biogenesis

Stimulator of interferon genes (STING) is activated in many pathophysiological conditions, leading to TBK1-dependent interferon production in higher organisms. However, primordial functions of STING independent of TBK1 are poorly understood. Here, through proteomics and bioinformatics approaches, we identify lysosomal biogenesis as an unexpected function of STING. Transcription factor EB (TFEB), an evolutionarily conserved regulator of lysosomal biogenesis and host defense, is activated by STING from multiple species, including humans, mice, and frogs. STING-mediated TFEB activation is independent of TBK1, but it requires STING trafficking and its conserved proton channel. GABARAP lipidation, stimulated by the channel of STING, is key for STING-dependent TFEB activation. STING stimulates global upregulation of TFEB-target genes, mediating lysosomal biogenesis and autophagy. TFEB supports cell survival during chronic sterile STING activation, a common condition in aging and age-related diseases. These results reveal a primordial function of STING in the biogenesis of lysosomes, essential organelles in immunity and cellular stress resistance.

EB-SUN, a New Microtubule Plus-End Tracking Protein in Drosophila.

Microtubule (MT) regulation is essential for oocyte development. In Drosophila, MT stability, polarity, abundance, and orientation undergo dynamic changes across developmental stages. In our effort to identify novel microtubule-associated proteins (MAPs) that regulate MTs in the Drosophila ovary, we identified a previously uncharacterized gene, CG18190, encoding a novel MT end-binding (EB) protein, which we propose to name EB-SUN. We show that EB-SUN colocalizes with EB1 at growing microtubule plus-ends in Drosophila S2 cells. Tissue-specific and developmental expression profiles from Paralog Explorer reveal that EB-SUN is predominantly expressed in the ovary and early embryos, while EB1 is ubiquitously expressed. Furthermore, as early as oocyte determination, EB-SUN comets are highly concentrated in oocytes during oogenesis. EB-SUN knockout (KO) results in a decrease in MT density at the onset of mid-oogenesis (Stage 7) and delays oocyte growth during late mid-oogenesis (Stage 9). Combining EB-SUN KO with EB1 knockdown (KD) in germ cells significantly further reduced MT density at Stage 7. Notably, all eggs from EB-SUN KO/EB1 KD females fail to hatch, unlike single gene depletion, suggesting a functional redundancy between these two EB proteins during embryogenesis. Our findings indicate that EB-SUN and EB1 play distinct roles during early embryogenesis.

The food-borne carcinogenic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) disrupts circadian rhythms and ameliorated by pterostilbene (PSB) in Caenorhabditis elegans.

The food-borne 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potential human carcinogen abundant in cooked meat. While circadian rhythms are crucial biological oscillations, the negative impact of PhIP on circadian systems and the potential of mitigation remain underexplored. We investigated the effects of PhIP on circadian rhythms and the mitigating effects of the phytochemical antioxidant pterostilbene (PSB) in Caenorhabditis elegans. We show that exposure to 10μM PhIP disrupts the 24-h circadian rhythms of C. elegans, an effect mitigated by co-exposure to 100μM PSB. In addition, PhIP-induced circadian disruption can be linked to defective oxidative stress resistance, which is associated with the DAF-16/FOXO pathway and is modulated by PSB. Molecular docking suggested that PhIP and PSB bind similarly to DAF-16. Moreover, 10μM PhIP abolished the rhythmic expression of the core clock gene prdx-2, which is restored by 100μM PSB. Findings from this study provide novel insight of how food-borne contaminant like PhIP may contribute to the disruption of circadian rhythms and suggest potential for PSB to mitigate these effects in higher organisms.

Intrinsic Disorder and Other Malleable Arsenals of Evolved Protein Multifunctionality.

Microscopic evolution at the functional biomolecular level is an ongoing process. Leveraging functional and high-throughput assays, along with computational data mining, has led to a remarkable expansion of our understanding of multifunctional protein (and gene) families over the past few decades. Various molecular and intermolecular mechanisms are now known that collectively meet the cumulative multifunctional demands in higher organisms along an evolutionary path. This multitasking ability is attributed to a certain degree of intrinsic or adapted flexibility at the structure-function level. Evolutionary diversification of structure-function relationships in proteins highlights the functional importance of intrinsically disordered proteins/regions (IDPs/IDRs) which are highly dynamic biological soft matter. Multifunctionality is favorably supported by the fluid-like shapes of IDPs/IDRs, enabling them to undergo disorder-to-order transitions upon binding to different molecular partners. Other new malleable members of the protein superfamily, such as those involved in fold-switching, also undergo structural transitions. This new insight diverges from all traditional notions of functional singularity in enzyme classes and emphasizes a far more complex, multi-layered diversification of protein functionality. However, a thorough review in this line, focusing on flexibility and function-driven structural transitions related to evolved multifunctionality in proteins, is currently missing. This review attempts to address this gap while broadening the scope of multifunctionality beyond single protein sequences. It argues that protein intrinsic disorder is likely the most striking mechanism for expressing multifunctionality in proteins. A phenomenological analogy has also been drawn to illustrate the increasingly complex nature of modern digital life, driven by the need for multitasking, particularly involving media.

Automated Assignment of 15N And 13C Enrichment Levels in Doubly-Labeled Proteins.

Uniform enrichment of 15N and 13C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli, a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13C and 15N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15N and 13C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [15N,13C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [15N,13C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae.

The C-terminal 4CXXC-type zinc finger domain of CDCA7 recognizes hemimethylated DNA and modulates activities of chromatin remodeling enzyme HELLS.

The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites.

Forward genetic screen of the C. elegans million mutation library reveals essential, cell-autonomous contributions of BBSome proteins to dopamine signaling.

The nematode Caenorhabditis elegans is well known for its ability to support forward genetic screens to identify molecules involved in neuronal viability and signaling. The proteins involved in C. elegans dopamine (DA) regulation are highly conserved across evolution, with prior work demonstrating that the model can serve as an efficient platform to identify novel genes involved in disease-associated processes. To identify novel players in DA signaling, we took advantage of a recently developed library of pre-sequenced mutant nematodes arising from the million mutation project (MMP) to identify strains that display the DA-dependent swimming-induced-paralysis phenotype (Swip). Our screen identified novel mutations in the dopamine transporter encoding gene dat-1, whose loss was previously used to identify the Swip phenotype, as well as multiple genes with previously unknown connections to DA signaling. Here, we present our isolation and characterization of one of these genes, bbs-1, previously linked to the function of primary cilia in worms and higher organisms, including humans, and where loss-of-function mutations result in a human disorder known as Bardet-Biedl syndrome. Our studies of C. elegans BBS-1 protein, as well as other proteins that are known to be assembled into a higher order complex (the BBSome) reveal that functional or structural disruption of this complex leads to exaggerated C. elegans DA signaling to produce Swip via a cell-autonomous mechanism. We provide evidence that not only does the proper function of cilia in C. elegans DA neurons support normal swimming behavior, but also that bbs-1 maintains normal levels of DAT-1 trafficking or function via a RHO-1 and SWIP-13/MAPK-15 dependent pathway where mutants may contribute to Swip independent of altered ciliary function. Together, these studies demonstrate novel contributors to DA neuron function in the worm and demonstrate the utility and efficiency of forward genetic screens using the MMP library.