Gene-specific Manner Research Articles

Tissue-Wide Effects Override Cell-Intrinsic Gene Function in Radial Neuron Migration.

The mammalian neocortex is composed of diverse neuronal and glial cell classes that broadly arrange in six distinct laminae. Cortical layers emerge during development and defects in the developmental programs that orchestrate cortical lamination are associated with neurodevelopmental diseases. The developmental principle of cortical layer formation depends on concerted radial projection neuron migration, from their birthplace to their final target position. Radial migration occurs in defined sequential steps, regulated by a large array of signaling pathways. However, based on genetic loss-of-function experiments, most studies have thus far focused on the role of cell-autonomous gene function. Yet, cortical neuron migration in situ is a complex process and migrating neurons traverse along diverse cellular compartments and environments. The role of tissue-wide properties and genetic state in radial neuron migration is however not clear. Here we utilized mosaic analysis with double markers (MADM) technology to either sparsely or globally delete gene function, followed by quantitative single-cell phenotyping. The MADM-based gene ablation paradigms in combination with computational modeling demonstrated that global tissue-wide effects predominate cell-autonomous gene function albeit in a gene-specific manner. Our results thus suggest that the genetic landscape in a tissue critically affects the overall migration phenotype of individual cortical projection neurons. In a broader context, our findings imply that global tissue-wide effects represent an essential component of the underlying etiology associated with focal malformations of cortical development in particular, and neurological diseases in general.

Изоформы микроРНК miR-148a и miR-203a предположительно играют роль супрессоров колоректального рака

MicroRNAs are short non-coding molecules which regulate translation in a gene-specific manner. MicroRNA isoforms that differ by few extra or missing nucleotides at the 5'-terminus (5'-isomiR) show strikingly different target specificity. This study aimed to identify functional roles of 5′-isomiR in colorectal cancers. Transcriptomic targets of microRNA isoforms were predicted using bioinformatics tools miRDB and TargetScan. The sets of putative targets identified for 5′-isomiR were integrated with mRNA and microRNA sequencing data for primary colorectal tumors retrieved from The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) database. The network of interactions among miRNA, their targets and transcription factors was built using the miRGTF-net algorithm. The results indicate that microRNA isoforms highly expressed in colorectal cancer and differing by a single nucleotide position at the 5'-terminus have ≤ 30% common targets. The regulatory network of interactions enables identification of the most engaged microRNA isoforms. Anti-correlated expression levels of canonical microRNA hsa-miR-148a-3p and its putative targets including CSF1, ETS1, FLT1, ITGA5, MEIS1, MITF and RUNX2 proliferation regulators suggest an anti-tumor role for this molecule. The canonical microRNA hsa-miR-203a-3p|0 and its 5′-isoform bind different sets of anti-correlated putative targets, although both of them interact with genes involved in the epithelial-mesenchymal transition: SNAI2 and TNC.

Shaping the Innate Immune Response Through Post-Transcriptional Regulation of Gene Expression Mediated by RNA-Binding Proteins.

Innate immunity is the frontline of defense against infections and tissue damage. It is a fast and semi-specific response involving a myriad of processes essential for protecting the organism. These reactions promote the clearance of danger by activating, among others, an inflammatory response, the complement cascade and by recruiting the adaptive immunity. Any disequilibrium in this functional balance can lead to either inflammation-mediated tissue damage or defense inefficiency. A dynamic and coordinated gene expression program lies at the heart of the innate immune response. This expression program varies depending on the cell-type and the specific danger signal encountered by the cell and involves multiple layers of regulation. While these are achieved mainly via transcriptional control of gene expression, numerous post-transcriptional regulatory pathways involving RNA-binding proteins (RBPs) and other effectors play a critical role in its fine-tuning. Alternative splicing, translational control and mRNA stability have been shown to be tightly regulated during the innate immune response and participate in modulating gene expression in a global or gene specific manner. More recently, microRNAs assisting RBPs and post-transcriptional modification of RNA bases are also emerging as essential players of the innate immune process. In this review, we highlight the numerous roles played by specific RNA-binding effectors in mediating post-transcriptional control of gene expression to shape innate immunity.

Complexities in the role of acetylation dynamics in modifying inducible gene activation parameters.

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous activation kinetics of a given gene in response to EGF stimulation. Acetylation levels modify these heterogenous patterns and influence both allele activation frequencies and overall expression profile parameters. Our data therefore point to a complex interplay between acetylation equilibria and target gene induction where acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner.

Integrative analysis of epigenetics data identifies gene-specific regulatory elements.

Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water.

Epigenetic approaches to crop breeding: current status and perspectives.

In order to tackle the cumulative adverse effects of global climate change, reduced farmland, and heightened needs of an ever-increasing world population, modern agriculture is in urgent search of solutions that can ensure world food security and sustainable development. Classical crop breeding is still a powerful method to obtain crops with valued agronomical traits, but its potential is gradually being compromised by the menacing decline of genetic variation. Resorting to the epigenome as a source of variation could serve as a promising alternative. Here, we discuss current status of epigenetics-mediated crop breeding (epibreeding), highlight its advances and limitations, outline currently available methodologies, and propose novel RNA-based strategies to modify the epigenome in a gene-specific and transgene-free manner.

From Antisense RNA to RNA Modification: Therapeutic Potential of RNA-Based Technologies.

Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2′-O-methylation.

Microenvironment controls inflammatory response in macrophages

Abstract Inflammatory response often changes tissue environment, such as pH, oxygen level or composition of cells. Sustained deviation in these variables from homeostasis leads to tissue dysfunction and pathology, as collateral damage of inflammation. Meanwhile, we begin to realized that immune cells also constantly monitor tissue environment and maintain tissue homeostasis. This raises an intriguing question: do changes in tissue environment feedback to modulate immune functions during inflammation? pH is normally maintained within a narrow homeostatic range. Decrease in pH, or known as acidosis, is associated with diverse inflammatory conditions. Using macrophages, we found that acidic pH modulates their inflammatory response to lipopolysaccharide (LPS, TLR4 agonist) in a gene-specific manner, independent of known pH-sensing receptors. Employing a computational deconvolution algorithm, we systemically characterized how change in pH and activation of TLR4 regulate inflammatory gene expression. This revealed three distinct gene classes: a group insensitive to environment, and two groups representing alternative inflammatory programs, whose control by LPS is either antagonistic or synergistic with acidic pH. Surprisingly, this influence by pH correlates with differential control by NF-kB and IRF3, but is not due to difference in pathway activity. Through profiling chromatin landscape, we discovered that pH regulates gene expression via the activity of specific enhancers and chromatin remodeling. Our findings highlight a critical role of tissue microenvironment in controlling inflammatory gene expression, and reveal qualitatively different responses encoded by transcription circuits and epigenetic mechanisms.

A collection of toolkit strains reveals distinct localization and dynamics of membrane-associated transcripts in epithelia.

Visualizing mRNA in real time invivo at high resolution is critical for a full understanding of the spatiotemporal dynamics of gene regulation and function. Here, using a PP7/PCP-based mRNA-tagging approach, we construct a collection of tissue-specific and differentially expressed toolkit strains for visualizing mRNAs encoding apical, basolateral, and junctional proteins in Caenorhabditis elegans epithelia. We precisely delineate the spatiotemporal organization and dynamics of these transcripts across multiple subcellular compartments and tissues. Remarkably, all the transcripts exhibit an asymmetric, membrane-associated localization during epithelial polarization and maturation, which suggests that mRNA localization is a prerequisite for epithelial polarization and function. Single-particle tracking reveals striking features of the transport dynamics of the mRNAs in a gene-specific, compartment-linked, and time-resolved manner. The toolkit can be used to identify the cis-regulatory elements and trans-acting factors for mRNA localization. This study provides a valuable resource to investigate complex RNA dynamics in epithelial polarity and morphogenesis.

ERN1 knockdown modifies the impact of glucose and glutamine deprivations on the expression of EDN1 and its receptors in glioma cells.

Objective. The aim of the present investigation was to study the impact of glucose and gluta-mine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in response to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up-regulated in control U87 glioma cells exposure under glucose deprivation condition in comparison with the glioma cells, growing in regular glucose containing medium. We also observed up-regulation of ECE1 gene expression in U87 glioma cells exposure under glutamine deprivation as well as down-regulation of the expression of EDN1 and EDNRA mRNA, being more significant for EDN1. Furthermore, the knockdown of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose and glutamine deprivation conditions. Thus, the ERN1 knockdown led to a strong suppression of EDN1 gene expression under glucose deprivation, but did not change the effect of glutamine deprivation on its expression. At the same time, the knockdown of ERN1 signaling introduced the sensitivity of EDNRB gene to both glucose and glutamine deprivations as well as completely removed the impact of glucose deprivation on the expression of ECE1 gene. Conclusions. The results of this study demonstrated that the expression of endothelin-1, its receptors, and ECE1 genes is preferentially sensitive to glucose and glutamine deprivations in gene specific manner and that knockdown of ERN1 significantly modified the expression of EDN1, EDNRB, and ECE1 genes in U87 glioma cells. It is possible that the observed changes in the expression of studied genes under nutrient deprivation may contribute to the suppressive effect of ERN1 knockdown on glioma cell proliferation and invasiveness.

BCOR modulates transcriptional activity of a subset of glucocorticoid receptor target genes involved in cell growth and mobility

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.

Gene- and organ-specific impact of paracetamol on Solanun nigrum L.’s γ-glutamylcysteine synthetase and glutathione S-transferase and consequent phytoremediation fitness

Paracetamol is considered a priority pharmaceutical because its presence in the environment may have an impact on human health and in food chains. To remediate this problem, the use of Solanum nigrum L. plants for the clean-up of paracetamol-contaminated sites was investigated. The study revealed that this plant species can be used as a remarkable paracetamol phytoremediation instrument, thus delivering a cost-effective and eco-friendly cleanup technology for its environmental removal. Paracetamol was not toxic to plants, exerting a growth-promoting effect up to 0.5 mg L−1. Plant tolerance mechanisms resided essentially on root GST-mediated conjugation of paracetamol to GSH, powered by the γ-ECS increased activity that locally provided the GSH needed. Increased activities for GST and γ–ECS were detected in roots and shoots of treated plants and these enzymes were regulated in an organ- and gene-specific manner: at the transcription level in roots and post-transcriptionally in shoots, with GSTs of the classes Theta and Phi playing the most relevant role in the plant detoxification process.

Abstract PS18-16: The complete estrogen receptor antagonist OP-1250 shrinks tumors in xenograft models and has favorable preclinical pharmacokinetic attributes

Abstract Antiestrogens are widely used to treat ER+, HER2- breast cancer however these may produce estrogen-like agonist effects in a cell and gene-specific manner. Furthermore, this partial agonism has been implicated in the development of tumor resistance. In contrast, a complete ER antagonist lacks agonist activity in all tissues. Given the high risk of recurrence in ER+ patients treated with endocrine therapies, we hypothesize that drugs that completely antagonize ER-mediated signaling will have a clinical advantage in treating resistant tumors, particularly in tumors for which the ER has acquired mutations conferring estrogen-independent growth. We previously identified OP-1250 in our laboratory as a complete antagonist and degrader of wild type and mutant ERalpha in breast and uterus cells. Here we explore the pharmacokinetic properties of orally administered OP-1250 and its ability to shrink breast tumors from xenograft models expressing both wild type and mutant estrogen receptors. Orally administered OP-1250 produced high and stable levels in multiple species, suggesting that once daily dosing may be sufficient to produce excellent drug exposure in patients. OP-1250 shrank ER+ tumors in multiple patient-derived tumor xenografts in mice at daily doses of 3 mg/kg, including in tumors expressing the Y537S allele of ERalpha that confers estrogen-independent growth and tumor resistance. Furthermore, OP-1250 accumulated within tumor specimens, indicating excellent target penetration was achieved with low daily doses. These preclinical studies indicate that OP-1250 has the potential to be a best-in-class antiestrogen due to its ability to complete antagonize ER, potent efficacy in shrinking tumors, and robust drug exposure. A Phase 1/2 dose escalation and expansion study in patients with ER+, HER2- metastatic or advanced BC previously treated with an endocrine therapy will be initiated this year. Citation Format: Leslie Hodges-Gallagher, Richard Sun, David C Myles, Pamela M Klein, Jo Anne Zujewski, Cyrus L Harmon, Peter J Kushner. The complete estrogen receptor antagonist OP-1250 shrinks tumors in xenograft models and has favorable preclinical pharmacokinetic attributes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-16.

Challenging the Integrity of Rhythmic Maternal Signals Revealed Gene-Specific Responses in the Fetal Suprachiasmatic Nuclei.

During fetal stage, maternal circadian system sets the phase of the developing clock in the suprachiasmatic nuclei (SCN) via complex pathways. We addressed the issue of how impaired maternal signaling due to a disturbed environmental light/dark (LD) cycle affects the fetal SCN. We exposed pregnant Wistar rats to two different challenges – a 6-h phase shift in the LD cycle on gestational day 14, or exposure to constant light (LL) throughout pregnancy – and detected the impact on gene expression profiles in 19-day-old fetuses. The LD phase shift, which changed the maternal SCN into a transient state, caused robust downregulation of expression profiles of clock genes (Per1, Per2, and Nr1d1), clock-controlled (Dbp) genes, as well as genes involved in sensing various signals, such as c-fos and Nr3c1. Removal of the rhythmic maternal signals via exposure of pregnant rats to LL abolished the rhythms in expression of c-fos and Nr3c1 in the fetal SCN. We identified c-fos as the gene primarily responsible for sensing rhythmic maternal signals because its expression profile tracked the shifted or arrhythmic maternal SCN clock. Pathways related to the maternal rhythmic behavioral state were likely not involved in driving the c-fos expression rhythm. Instead, introduction of a behavioral rhythm to LL-exposed mothers via restricted feeding regime strengthened rhythm in Vip expression in the fetal SCN. Our results revealed for the first time that the fetal SCN is highly sensitive in a gene-specific manner to various changes in maternal signaling due to disturbances of environmental cycles related to the modern lifestyle in humans.

Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes.

The FACT (FAcilitates Chromatin Transactions) complex influences transcription initiation and enables passage of RNA polymerase (Pol) II through gene body nucleosomes during elongation. In the budding yeast, ∼280 noncoding RNA genes highly transcribed in vivo by Pol III are found in the nucleosome-free regions bordered by positioned nucleosomes. The downstream nucleosome dynamics was found to regulate transcription via controlling the gene terminator accessibility and hence, terminator-dependent Pol III recycling. As opposed to the enrichment at the 5′-ends of Pol II-transcribed genes, our genome-wide mapping found transcription-dependent enrichment of the FACT subunit Spt16 near the 3′-end of all Pol III-transcribed genes. Spt16 physically associates with the Pol III transcription complex and shows gene-specific occupancy levels on the individual genes. On the non-tRNA Pol III-transcribed genes, Spt16 facilitates transcription by reducing the nucleosome occupany on the gene body. On the tRNA genes, it maintains the position of the nucleosome at the 3′ gene end and affects transcription in a gene-specific manner. Under nutritional stress, Spt16 enrichment is abolished in the gene downstream region of all Pol III-transcribed genes and reciprocally changed on the induced or repressed Pol II-transcribed ESR genes. Under the heat and replicative stress, its occupancy on the Pol III-transcribed genes increases significantly. Our results show that Spt16 elicits a differential, gene-specific and stress-responsive dynamics, which provides a novel stress-sensor mechanism of regulating transcription against external stress. By primarily influencing the nucleosomal organization, FACT links the downstream nucleosome dynamics to transcription and environmental stress on the Pol III-transcribed genes.

Basics and disturbances of genomic imprinting

Abstract Genomic imprinting ensures the parent-specific expression of either the maternal or the paternal allele, by different epigenetic processes (DNA methylation and histone modifications) that confer parent-specific marks (imprints) in the paternal and maternal germline, respectively. Most protein-coding imprinted genes are involved in embryonic growth, development, and behavior. They are usually organized in genomic domains that are regulated by differentially methylated regions (DMRs). Genomic imprints are erased in the primordial germ cells and then reset in a gene-specific manner according to the sex of the germline. The imprinted genes regulate and interact with other genes, consistent with the existence of an imprinted gene network. Defects of genomic imprinting result in syndromal imprinting disorders. To date a dozen congenital imprinting disorders are known. Usually, a given imprinting disorder can be caused by different types of defects, including point mutations, deletions/duplications, uniparental disomy, and epimutations. Causative trans-acting factors in imprinting disorders, including ZFP57 and the subcortical maternal complex (SCMC), have the potential to affect multiple DMRs across the genome, resulting in a multi-locus imprinting disturbance. There is evidence that mutations in components of the SCMC can confer an increased risk for imprinting disorders.

Prenatal Lead (Pb) Exposure’s Effect on DNA Methylation (5mC) and Hydroxymethylation (5hmC) in Adolescent Whole Blood

Gestational lead (Pb) exposure may adversely impact offspring health through epigenetic alterations via DNA methylation (5mC), addition of a methyl group to the 5th carbon of cytosine, and hydroxymethylation (5hmC), an intermediate in oxidative demethylation associated with increased gene expression. Most current methods collectively measure 5mC and 5hmC without distinguishing between the two. This study aims to identify the association of prenatal Pb exposure at each trimester (T1, T2, T3) with 5mC and 5hmC levels at multiple cytosine-phosphate-guanine (CpG) sites within candidate regions of genes previously associated with prenatal Pb exposure (HCN2, NINJ2, RAB5A, and TPPP) in blood leukocytes of children ages 11-18 years. Participants from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) birth cohorts were selected (n=144) for pyrosequencing analysis following oxidative or standard sodium bisulfite treatment. Participants are 51% male with concurrent blood lead levels (BLL) averaging 3.29±4.44µg/dL and historical maternal BLL averaging 6.43±5.16µg/dL (T1), 5.66±5.21µg/dL (T2), and 5.86±4.34µg/dL (T3). 5mC levels were quantified in HCN2 (mean±SD, 81.3±9.63%), NINJ2 (36.7±22.5%), RAB5A (1.40±1.20%), and TPPP (92.5±8.03%). 5hmC levels were estimated in HCN2 (2.04±4.30%), NINJ2 (2.15±5.43%), RAB5A (0.64±0.80%), and TPPP (0.45±8.01%), providing evidence for 5hmC presence in blood leukocytes. Controlling for sex, current BLL, child’s age, and pyrosequencing batch, T1 maternal BLL was associated with 5mC in HCN2 (β=-1.256, p=0.019) and NINJ2 (β=0.426, p=0.004); T2 BLL with 5mC in HCN2 (β=0.368, p=0.028) and 5hmC in NINJ2 (β=0.232, p=0.011); and T3 BLL with 5mC in HCN2 (β=0.502, p=0.016) and 5hmC in NINJ2 (β=0.372, p=0.001). No associations were identified between Pb and RAB5A and TPPP. Together, these data suggest that perinatal Pb exposure results may alter 5mC and 5hmC in blood in a gene-specific manner that persists into adolescence.

Validation of prostate cancer risk variants rs10993994 and rs7098889 by CRISPR/Cas9 mediated genome editing.

GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the β-microseminoprotein (MSMB) promoter region, mediates MSMB prostate secretion levels, and is linked to mRNA expression changes in both MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in positive linkage disequilibrium with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation studies to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available. AUTHOR SUMMARY: In pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in the human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and cell-type specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded β-MSP in prostate cancer pathogenesis.

New Insights Into the Evolutionary History of Melatonin Receptors in Vertebrates, With Particular Focus on Teleosts.

In order to improve our understanding of melatonin signaling, we have reviewed and revised the evolutionary history of melatonin receptor genes (mtnr) in vertebrates. All gnathostome mtnr genes have a conserved gene organization with two exons, except for mtnr1b paralogs of some teleosts that show intron gains. Phylogeny and synteny analyses demonstrate the presence of four mtnr subtypes, MTNR1A, MTNR1B, MTNR1C, MTNR1D that arose from duplication of an ancestral mtnr during the vertebrate tetraploidizations (1R and 2R). In tetrapods, mtnr1d was lost, independently, in mammals, in archosaurs and in caecilian amphibians. All four mtnr subtypes were found in two non-teleost actinopterygian species, the spotted gar and the reedfish. As a result of teleost tetraploidization (3R), up to seven functional mtnr genes could be identified in teleosts. Conservation of the mtnr 3R-duplicated paralogs differs among the teleost lineages. Synteny analysis showed that the mtnr1d was conserved as a singleton in all teleosts resulting from an early loss after tetraploidization of one of the teleost 3R and salmonid 4R paralogs. Several teleosts including the eels and the piranha have conserved both 3R-paralogs of mtnr1a, mtnr1b, and mtnr1c. Loss of one of the 3R-paralogs depends on the lineage: mtnr1ca was lost in euteleosts whereas mtnr1cb was lost in osteoglossomorphs and several ostariophysians including the zebrafish. We investigated the tissue distribution of mtnr expression in a large range of tissues in medaka. The medaka has conserved the four vertebrate paralogs, and these are expressed in brain and retina, and, differentially, in peripheral tissues. Photoperiod affects mtnr expression levels in a gene-specific and tissue-specific manner. This study provides new insights into the repertoire diversification and functional evolution of the mtnr gene family in vertebrates.

Expression and subcellular analyses of CCR8a/b genes with the identification of response to SGIV viral infect in orange-spotted grouper (Epinephelus coioides)

Chemokine receptors are a superfamily of seven transmembrane domain G-coupled receptors, and they play important roles in immune surveillance, inflammation, and development. Recently, nine CC chemokine receptors (CCRs) were identified and cloned from orange-spotted grouper (Epinephelus coioides) and annotated by phylogenetic and syntenic analyses. We detected mRNA transcripts for CCRs in healthy tissues of E. coioides, and CCR genes were highly expressed in the immune-relevant tissues. Analysis of gene expression after Singapore grouper iridovirus (SGIV) infection indicated that CCR genes are regulated in a gene-specific manner. CCR8a and CCR8b were significantly upregulated in the spleen and liver of resistant fish, indicating potential roles in immunity against the pathogen. Fluorescence microscopy revealed that CCR8a and CCR8b were expressed predominantly in the cytoplasm. Overexpression of CCR8a and CCR8b in grouper cells significantly inhibited the replication of SGIV, demonstrating that they delayed the occurrence of cytopathic effects induced by SGIV infection and inhibited viral gene transcription. CCR8a and CCR8b overexpression also significantly increased the expression of interferon (IFN)-related cytokines and activated IFN response element and IFN promoter activities. These results demonstrated that CCR8a and CCR8b might have an antiviral function against SGIV infect.