ABSTRACT
Sunlight exposure is known to affect mood, learning, and cognition. However, the molecular and cellular mechanisms remain elusive. Here, we show that moderate UV exposure elevated blood urocanic acid (UCA), which then crossed the blood-brain barrier. Single-cell mass spectrometry and isotopic labeling revealed a novel intra-neuronal metabolic pathway converting UCA to glutamate (GLU) after UV exposure. This UV-triggered GLU synthesis promoted its packaging into synaptic vesicles and its release at glutamatergic terminals in the motor cortex and hippocampus. Related behaviors, like rotarod learning and object recognition memory, were enhanced after UV exposure. All UV-induced metabolic, electrophysiological, and behavioral effects could be reproduced by the intravenous injection of UCA and diminished by the application of inhibitor or short hairpin RNA (shRNA) against urocanase, an enzyme critical for the conversion of UCA to GLU. These findings reveal a new GLU biosynthetic pathway, which could contribute to some of the sunlight-induced neurobehavioral changes.
Subject(s)
Brain/radiation effects , Glutamic Acid/biosynthesis , Learning/radiation effects , Memory/radiation effects , Ultraviolet Rays , Animals , Brain/metabolism , Brain/pathology , Chromatography, High Pressure Liquid , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Neurons/cytology , Neurons/physiology , Patch-Clamp Techniques , RNA Interference , RNA, Small Interfering/metabolism , Tandem Mass Spectrometry , Urocanate Hydratase/antagonists & inhibitors , Urocanate Hydratase/genetics , Urocanate Hydratase/metabolism , Urocanic Acid/blood , Urocanic Acid/metabolismABSTRACT
Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.
Subject(s)
Enhancer Elements, Genetic , Estrogen Receptor alpha/metabolism , Transcription Factors/metabolism , Estrogens/metabolism , GATA3 Transcription Factor/metabolism , Gene Expression Regulation , Hepatocyte Nuclear Factor 3-alpha/metabolism , Humans , Multiprotein Complexes/metabolismABSTRACT
Enhancer-promoter communication is known to regulate spatiotemporal dynamics of gene expression. Several methods are available to capture enhancer-promoter interactions, but they either require large amounts of starting materials and are costly, or provide a relative low resolution in chromatin contact maps. Here, we present nicking enzyme-assisted open chromatin interaction capture (NicE-C), a method that leverages nicking enzyme-mediated open chromatin profiling and chromosome conformation capture to enable robust and cost-effective detection of open chromatin interactions at high resolution, especially enhancer-promoter interactions. Using TNF stimulation and mouse kidney aging as models, we applied NicE-C to reveal characteristics of dynamic enhancer-promoter interactions.
Subject(s)
Chromatin , Enhancer Elements, Genetic , Animals , Chromatin/genetics , Chromosomes/genetics , Mice , Promoter Regions, GeneticABSTRACT
Lung cancer is a major cause accounting for cancer-related mortalities, with lung adenocarcinoma (LUAD) being the most prevalent subtype. Given the high clinical and cellular heterogeneities of LUAD, accurate diagnosis and prognosis are crucial to avoid overdiagnosis and overtreatment. Taking full advantage of scRNA-Seq data to resolve the tumor heterogeneities, we explored the overall landscape of LUAD microenvironment. Utilizing the stage-specific tumor cell markers, we have developed highly accurate diagnostic and prognostic models with elevated sensitivity and specificity. The diagnostic model, developed through random forest algorithms with a thirteen-gene signature, achieved an accuracy of 96.4% and an AUC of 0.993. These metrics were further demonstrated by benchmarking with available models and scoring systems in independent cohorts. Concurrently, the prognostic model, formulated via Cox regression with a six-gene signature, effectively predicted overall survival, with elevated risk scores associated with increased fractions of cancer-associated fibroblasts, and higher likelihood of immune escape and T-cell exclusion. Subsequently, two nomograms were developed to predict survival and drug responses, facilitating their integration into clinical practice. Overall, this study underscores the potential of our models for efficient, rapid, and cost-effective diagnosis and prognosis of LUAD, adaptable to multiple expression profiling platforms and quantification methods.
ABSTRACT
BACKGROUND: Homology-based recombination (HR) is the cornerstone of genetic mapping. However, a lack of sufficient sequence homology or the presence of a genomic rearrangement prevents HR through crossing, which inhibits genetic mapping in relevant genomic regions. This is particularly true in species hybrids whose genomic sequences are highly divergent along with various genome arrangements, making the mapping of genetic loci, such as hybrid incompatibility (HI) loci, through crossing impractical. We previously mapped tens of HI loci between two nematodes, Caenorhabditis briggsae and C. nigoni, through the repeated backcrossing of GFP-linked C. briggsae fragments into C. nigoni. However, the median introgression size was over 7 Mb, indicating apparent HR suppression and preventing the subsequent cloning of the causative gene underlying a given HI phenotype. Therefore, a robust method that permits recombination independent of sequence homology is desperately desired. RESULTS: Here, we report a method of highly efficient targeted recombination (TR) induced by CRISPR/Cas9 with dual guide RNAs (gRNAs), which circumvents the HR suppression in hybrids between the two species. We demonstrated that a single gRNA was able to induce efficient TR between highly homologous sequences only in the F1 hybrids but not in the hybrids that carry a GFP-linked C. briggsae fragment in an otherwise C. nigoni background. We achieved highly efficient TR, regardless of sequence homology or genetic background, when dual gRNAs were used that each specifically targeted one parental chromosome. We further showed that dual gRNAs were able to induce efficient TR within genomic regions that had undergone inversion, in which HR-based recombination was expected to be suppressed, supporting the idea that dual-gRNA-induced TR can be achieved through nonhomology-based end joining between two parental chromosomes. CONCLUSIONS: Recombination suppression can be circumvented through CRISPR/Cas9 with dual gRNAs, regardless of sequence homology or the genetic background of the species hybrid. This method is expected to be applicable to other situations in which recombination is suppressed in interspecies or intrapopulation hybrids.
Subject(s)
Caenorhabditis , Animals , Caenorhabditis/genetics , CRISPR-Cas Systems , Chromosome Mapping , Genome , Recombination, GeneticABSTRACT
BACKGROUND: SAHA was reported to enhance the expression of miR-129-5p, which was predicted to bind to 3' UTR of CASP-6, a gene playing crucial roles in the pathogenesis of memory impairment. Whether SAHA/miR-129-5p/CASP-6 is involved in the pathogenesis of prenatal exposure to sevoflurane remains to be explored. METHODS: Morris water maze test was performed to evaluate the functional parameters of learning and memory. Quantitative real-time qPCR was carried out to analyze the expression of miRNAs and CASP-6 mRNA under different conditions. RESULTS: Sevoflurane exposure of pregnant rats and SAHA treatment of the offspring had no effect on the blood gases, litter size, survival rate and weight. SAHA administration remarkably reversed the learning and memory impairment in prenatal rats caused by sevoflurane exposure. Mechanistically, the abnormal expression of miR-129-5p and CASP-6 in the offspring of pregnant rats exposed to sevoflurane was effectively restored by SAHA treatment. The luciferase activity of CASP-6 vector was effectively inhibited by miR-129-5p in primary neuron cells of rats. Moreover, the expression of CASP-6 mRNA and protein was significantly suppressed by miR-129-5p and SAHA treatment in a dose-dependent manner. CONCLUSION: Our work demonstrated that the administration of SAHA suppressed the expression of CASP-6 via modulating the expression of miR-129-5p, and SAHA may rescue the apoptosis of neurons caused by exposure to sevoflurane. The underlying mechanism might be the ability of SAHA to relieve learning and memory impairment in the offspring of the pregnant rats exposed to sevoflurane.
Subject(s)
Anesthesia , MicroRNAs , Pregnancy , Female , Rats , Animals , Sevoflurane/pharmacology , Vorinostat/pharmacology , Learning , Memory Disorders/chemically induced , Memory Disorders/drug therapy , Memory Disorders/metabolism , MicroRNAs/metabolism , Hippocampus/metabolismABSTRACT
The somatic macronucleus (MAC) and germline micronucleus (MIC) of Tetrahymena thermophila differ in chromosome numbers, sizes, functions, transcriptional activities, and cohesin complex location. However, the higher-order chromatin organization in T. thermophila is still largely unknown. Here, we explored the higher-order chromatin organization in the two distinct nuclei of T. thermophila using the Hi-C and HiChIP methods. We found that the meiotic crescent MIC has a specific chromosome interaction pattern, with all the telomeres or centromeres on the five MIC chromosomes clustering together, respectively, which is also helpful to identify the midpoints of centromeres in the MIC. We revealed that the MAC chromosomes lack A/B compartments, topologically associating domains (TADs), and chromatin loops. The MIC chromosomes have TAD-like structures but not A/B compartments and chromatin loops. The boundaries of the TAD-like structures in the MIC are highly consistent with the chromatin breakage sequence (CBS) sites, suggesting that each TAD-like structure of the MIC chromosomes develops into one MAC chromosome during MAC development, which provides a mechanism of the formation of MAC chromosomes during conjugation. Overall, we demonstrated the distinct higher-order chromatin organization in the two nuclei of the T. thermophila and suggest that the higher-order chromatin structures may play important roles during the development of the MAC chromosomes.
Subject(s)
Chromatin/chemistry , Chromosomes/chemistry , Macronucleus/genetics , Micronucleus, Germline/genetics , Tetrahymena thermophila/genetics , Centromere , Meiosis/geneticsABSTRACT
Enhancers instruct spatio-temporally specific gene expression in a manner tightly linked to higher-order chromatin architecture. Critical chromatin architectural regulators condensin I and condensin II play non-redundant roles controlling mitotic chromosomes. But the chromosomal locations of condensins and their functional roles in interphase are poorly understood. Here we report that both condensin complexes exhibit an unexpected, dramatic estrogen-induced recruitment to estrogen receptor α (ER-α)-bound eRNA(+) active enhancers in interphase breast cancer cells, exhibiting non-canonical interaction with ER-α via its DNA-binding domain (DBD). Condensins positively regulate ligand-dependent enhancer activation at least in part by recruiting an E3 ubiquitin ligase, HECTD1, to modulate the binding of enhancer-associated coactivators/corepressors, including p300 and RIP140, permitting full eRNA transcription, formation of enhancer:promoter looping, and the resultant coding gene activation. Collectively, our results reveal an important, unanticipated transcriptional role of interphase condensins in modulating estrogen-regulated enhancer activation and coding gene transcriptional program.
Subject(s)
Adenosine Triphosphatases/metabolism , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Estrogen Receptor alpha/metabolism , Multiprotein Complexes/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Base Sequence , Binding Sites , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Chromatin/genetics , Chromatin/metabolism , DNA, Neoplasm/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Estradiol/metabolism , Female , Gene Knockdown Techniques , Humans , Interphase , MCF-7 Cells , Models, Biological , Molecular Sequence Data , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/genetics , Nuclear Proteins/metabolism , Nuclear Receptor Interacting Protein 1 , Promoter Regions, Genetic , Protein Binding , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Adenylate kinase 6 (AK6), a nucleus localized phosphotransferase in mammalians, shows ubiquitously expression and broad substrate activity in different tissues and cell types. Although the function of AK6 has been extensively studied in different cancer cell lines, its role in mammalian germline is still unknown. Here we showed that knockdown of AK6 inhibits cell proliferation and promotes cell apoptosis in human testicular carcinoma (NT2 cells). Co-immunoprecipitation experiment and in vitro pull down assay identified WNK1 (with no lysine kinase-1) as one of the AK6 interacting proteins in NT2 cells. Moreover, we found that AK6 regulates the phosphorylation states of WNK1 (Thr60) and affects phosphorylation level of Akt (Ser473) upon hypotonic condition, probably affecting chloride channel and regulating ion transport and homeostasis in NT2 cells and consequently contributing to the decreased cell proliferation rate. In conclusion, AK6 regulates WNK1 phosphorylation states and affects ion homeostasis in NT2 cells. These findings provide new insights into the function of AK6 and WNK1 in human testicular carcinoma. This work also provides foundation for further mechanism study of AK6 in spermatogenesis.
Subject(s)
Adenylate Kinase/genetics , Carcinoma/genetics , Cell Proliferation/genetics , Testicular Neoplasms/genetics , Apoptosis/genetics , Carcinoma/pathology , Cell Line, Tumor , Homeostasis/genetics , Humans , Male , Phosphorylation/genetics , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction/genetics , Testicular Neoplasms/pathology , WNK Lysine-Deficient Protein Kinase 1/geneticsABSTRACT
Long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) is nuclear-located and transcribed from chromatin 11. To date, little is known about the cellular functions and regulatory mechanisms of NEAT1 in prostate cancer (PCa). In this study, whole-genome RNA sequencing data were downloaded from TCGA and GEO databases. Biological information was used to analyze the different expressions of NEAT1. In situ hybridization (ISH) was performed to detect the expression of NEAT1 in PCa and paracarcinoma clinical samples. Then, NEAT1 was knocked down in PC3 cells through lentiviral infection with a plasmid construct. Bioinformatics and integrative analytical approaches were utilized to identify the relationships of NEAT1 with specific cancer-related gene sets. Cell proliferation assay and colony formation assay were performed to evaluate the cell proliferative ability. Glycolysis stress test, metabolism assay, and infiltrating T-cell function analysis were implemented to assess the changes in metabolism and immune microenvironment of PCa. We found that the expression of NEAT1 was higher in PCa than in non-neoplastic tissues. The cell proliferative capability of PCa cells was significantly reduced in the NEAT1 knockdown group. PCR array and bioinformatics analysis revealed that the enrichment of acidic substance-related gene sets was associated with NEAT1 expression. NEAT1 depletion inhibited PCa cell aerobic glycolysis accompanied by the reduction of lactate levels in the medium. Further, we found that lactate dehydrogenase A (LDHA) expression was positively regulated by NEAT1. At last, co-culture systems indicated that NEAT1 or LDHA knockdown promoted the secretion of CD8+ T-lymphocyte factors, including TNF-α, IFN-γ, and Granzyme B, and enhanced the antitumor effects.
Subject(s)
Immunologic Surveillance , MicroRNAs , Prostatic Neoplasms , RNA, Long Noncoding , T-Lymphocytes , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Glycolysis/genetics , Humans , Male , MicroRNAs/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Long Noncoding/genetics , T-Lymphocytes/immunology , Tumor MicroenvironmentABSTRACT
Long noncoding RNAs (lncRNAs) represent a group of regulatory RNAs that play critical roles in numerous cellular events, but their functional importance in development remains largely unexplored. Here, we discovered a series of previously unidentified gene clusters harboring conserved lncRNAs at the nonimprinting regions in brain (CNIBs). Among the seven identified CNIBs, human CNIB1 locus is located at Chr 9q33.3 and conserved from Danio rerio to Homo sapiens. Chr 9q33.3-9q34.11 microdeletion has previously been linked to human nail-patella syndrome (NPS) which is frequently accompanied by developmental and visual deficiencies. By generating CNIB1 deletion alleles in zebrafish, we demonstrated the requirement of CNIB1 for proper growth and development, and visual activities. Furthermore, we found that the role of CNIB1 on visual activity is mediated through a regulator of ocular development-lmx1bb. Collectively, our study shows that CNIB1 lncRNAs are important for zebrafish development and provides an lncRNA cluster-mediated pathophysiological mechanism for human Chr 9q33.3-9q34.11 microdeletion syndrome.
Subject(s)
RNA, Long Noncoding/genetics , Vision, Ocular/genetics , Animals , Brain/metabolism , Chromosome Deletion , Chromosomes, Human, Pair 9/genetics , Craniofacial Abnormalities/genetics , Genetic Loci , Genome , Heart Defects, Congenital/genetics , Humans , Intellectual Disability/genetics , Introns , Locomotion/genetics , Male , Mice, Inbred BALB C , RNA, Long Noncoding/metabolism , Transcription Factors/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/metabolismABSTRACT
Sperm proteins play vital roles in fertilization, but little is known about their identities in free-spawning marine invertebrates. Here, 286 sperm proteins are reported from the Hong Kong oyster Crassostrea hongkongensis using label-free and semi-quantitative proteomics. Proteins extracted from three sperm samples are separated by SDS-PAGE, analyzed by LC-MS/MS, and identified using Mascot. Functional classification of the sperm proteome reveals energy metabolism (33%), signaling and binding (23%), and protein synthesis and degradation (12%) as the top functional categories. Comparison of orthologous sperm proteins between C. hongkongensis, Crassostrea gigas, Mytilus edulis, and M. galloprovincialis suggests that energy metabolism (48%) is the most conserved functional group. Sequence alignment of the C. hongkongensis bindin, an acrosomal protein that binds the sperm and the egg, with those of three other Crassostrea species, reveals several conserved motifs. The study has enriched the data of invertebrate sperm proteins and may contribute to studies of mechanisms of fertilization in free-spawning invertebrates. The proteomic data are available in ProteomeXchange with the identifier PXD018255.
Subject(s)
Crassostrea , Proteome , Proteomics , Animals , Chromatography, Liquid , Male , Spermatozoa/physiology , Tandem Mass SpectrometryABSTRACT
Pancreatic cancer is a major cause of mortality with a poor diagnosis and prognosis that most often occurs in elderly patients. Few studies, however, focus on the interplay of age and pancreatic cancer at the transcriptional level. Here we evaluated the possible roles of age-dependent, differentially expressed genes (DEGs) in pancreatic cancer. These DEGs were used to construct a correlation network and clustered in six gene modules, among which two modules were highly correlated with patients' survival time. Integrating different datasets, including ATAC-Seq and ChIP-Seq, we performed multi-parallel analyses and identified eight age-dependent protein coding genes and two non-coding RNAs as potential candidates. These candidates, together with KLF5, a potent functional transcription factor in pancreatic cancer, are likely to be key elements linking cellular senescence and pancreatic cancer, providing insights on the balance between them, as well as on diagnosis and subsequent prognosis of pancreatic cancer.
Subject(s)
Aging/genetics , Biomarkers, Tumor , Gene Expression Regulation, Neoplastic , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , RNA, Long Noncoding/genetics , Cell Proliferation , Computational Biology/methods , Disease Progression , Gene Expression Profiling , Gene Regulatory Networks , Humans , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/therapy , Transcription Factors/metabolismABSTRACT
H2A.Y is an essential, divergent Tetrahymena thermophila histone variant. It has a long nonhistone N terminus that contains leucine-rich repeats (LRR) and an LRR cap domain with similarity to Sds22p, a regulator of yeast protein phosphatase 1 (PP1) activity in the nucleus. In growing cells, H2A.Y is incorporated into micronuclei only during S phase, which occurs immediately after micronuclear mitosis. Depletion of H2A.Y causes prolonged retention of mitosis-associated histone H3-S10 phosphorylation and mitotic abnormalities that mimic S10E mutation. In cells where H2A.Y is depleted, an inducible chimeric gene, in which the H2A.Y N terminus is attached to H2A.X, is shown to regulate micronuclear H3-S10 phosphorylation. H2A.Y can also be specifically coimmunoprecipitated with a Tetrahymena PP1 ortholog (Ppo1p). Taken together, these results argue that the N terminus of H2A.Y functions to regulate H3-S10 dephosphorylation. This striking in vivo case of "cross-talk" between a H2A variant and a specific post-translational modification of another histone demonstrates a novel function for a histone variant.
Subject(s)
Histones/metabolism , Mitosis , Mutant Chimeric Proteins/metabolism , Protein Processing, Post-Translational , Tetrahymena thermophila/cytology , Tetrahymena thermophila/metabolism , Amino Acid Sequence , DNA Replication , Gene Knockout Techniques , Histones/classification , Histones/genetics , Molecular Sequence Data , Mutant Chimeric Proteins/classification , Mutant Chimeric Proteins/genetics , Nucleosomes/metabolism , Phosphorylation , Phylogeny , Protein Phosphatase 1/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/metabolism , ras Proteins/metabolismABSTRACT
The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17ß-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.
Subject(s)
Enhancer Elements, Genetic/genetics , Estrogens/pharmacology , RNA, Untranslated/genetics , Transcriptional Activation/drug effects , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Humans , Ligands , MCF-7 Cells , Nucleic Acid Conformation/drug effects , Promoter Regions, Genetic/genetics , RNA, Untranslated/biosynthesis , RNA, Untranslated/metabolism , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Transcriptional Activation/genetics , CohesinsABSTRACT
Antimicrobial resistance has been seriously threatening human health, and discovering new antimicrobial agents from the natural resource is still an important pathway among various strategies to prevent resistance. Guanidine-containing polyhydroxyl macrolides, containing a polyhydroxyl lactone ring and a guanidyl side chain, can be produced by many actinomycetes and have been proved to possess many bioactivities, especially broad-spectrum antibacterial and antifungal activities. To explore the potential of these compounds to be developed into new antimicrobial agents, a review on their structural diversities, spectroscopic characterizations, bioactivities, acute toxicities, antimicrobial mechanisms, and the structure-activity relationship was first performed based on the summaries and analyses of related publications from 1959 to 2019. A total of 63 guanidine-containing polyhydroxyl macrolides were reported, including 46 prototype compounds isolated from 33 marine and terrestrial actinomycetes and 17 structural derivatives. Combining with their antimicrobial mechanisms, structure-activity relationship analyses indicated that the terminal guanidine group and lactone ring of these compounds are vital for their antibacterial and antifungal activities. Further, based on their bioactivities and toxicity analyses, the discovery of guanidyl side-chain targeting to lipoteichoic acid of Staphylococcus aureus indicated that these compounds have a great potency to be developed into antimicrobial and anti-inflammatory drugs.
Subject(s)
Guanidine/chemistry , Guanidine/pharmacology , Macrolides/chemistry , Macrolides/pharmacology , Phylogeny , Spectrum Analysis , Structure-Activity Relationship , Toxicity Tests, AcuteABSTRACT
BACKGROUND: Excessive nerve growth factor (NGF) is commonly found in the follicular fluid of patients with polycystic ovary syndrome (PCOS). Furthermore, oocytes from PCOS patients exhibit lower developmental competence. The purpose of this study was to explore the association between excessive NGF and low oocyte competence in vitro. METHODS: Excessive NGF was added to mouse cumulus oocyte complexes (COCs) cultured in vitro to investigate meiotic maturation of the oocyte. After culture, mRNA expression levels of Pfkp and Ldha genes in cumulus cells (CCs) and Gdf9, Bmp15 and Fgf8 genes in oocytes, were determined by real-time quantitative polymerase chain reaction (qPCR). We also investigated the mRNA content of Pfkp and Ldha in CCs from PCOS and non-PCOS patients. RESULTS: Excessive NGF significantly inhibited oocyte meiotic maturation. The inhibitory effect was mediated by the NGF high-affinity receptor, NTRK1. mRNA content of Pfkp and Ldha genes in CCs was significantly reduced by excessive NGF stimulation. Moreover, the expression levels of Gdf9, Bmp15 and Fgf8 were also decreased in oocytes, and was induced by excessive NGF-stimulated CCs. In addition, lower expression levels of Pfkp and Ldha in CCs were identified in Chinese PCOS patients with excessive NGF (PCOS, 22 ± 2.63 ng/ml, n = 13; non-PCOS, 7.18 ± 2.42 ng/ml, n = 9; p < 0.01) in the follicular fluid, suggesting a potential association between excessive NGF and decreased glycolysis in the CCs of women with PCOS. CONCLUSIONS: Excessive NGF impairs bidirectional communication between oocyte and cumulus cells, which might be related to low oocyte competence.
Subject(s)
Cell Communication/drug effects , Cumulus Cells/physiology , Nerve Growth Factor/administration & dosage , Oocytes/physiology , Adult , Animals , Cells, Cultured , China , Cumulus Cells/chemistry , Dose-Response Relationship, Drug , Female , Follicular Fluid/chemistry , Glycolysis/drug effects , Humans , In Vitro Oocyte Maturation Techniques , Meiosis/drug effects , Mice , Mice, Inbred ICR , Nerve Growth Factor/analysis , RNA, Messenger/analysis , Receptor, trkA/analysisABSTRACT
Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type.
Subject(s)
Corticotrophs/physiology , DNA-Binding Proteins/physiology , Enhancer Elements, Genetic , LIM Domain Proteins/physiology , Promoter Regions, Genetic , Animals , Cell Line , DNA-Binding Proteins/metabolism , LIM Domain Proteins/metabolism , Mice , Mice, KnockoutABSTRACT
Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the 9p21 gene desert associated with coronary artery disease (CAD) and type 2 diabetes. Despite evidence for a role of the associated interval in neighbouring gene regulation, the biological underpinnings of these genetic associations with CAD or type 2 diabetes have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene desert for predicted enhancers and six times denser than the whole genome (P < 6.55 × 10(-33)). The CAD risk alleles of SNPs rs10811656 and rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines homozygous for the CAD risk haplotype show no binding of STAT1, and in lymphoblastoid cell lines homozygous for the CAD non-risk haplotype, binding of STAT1 inhibits CDKN2BAS (also known as CDKN2B-AS1) expression, which is reversed by short interfering RNA knockdown of STAT1. Using a new, open-ended approach to detect long-distance interactions, we find that in human vascular endothelial cells the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of IFNA21. In human vascular endothelial cells, interferon-γ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1-binding, long-range enhancer interactions and altered expression of neighbouring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signalling in a vascular cell type and thus demonstrate the utility of genome-wide association study findings in directing studies to novel genomic loci and biological processes important for disease aetiology.
Subject(s)
Chromosomes, Human, Pair 9/genetics , Coronary Artery Disease/genetics , Enhancer Elements, Genetic/genetics , Genetic Predisposition to Disease/genetics , Genetic Variation , Interferon-gamma/pharmacology , Signal Transduction/drug effects , Alleles , Cell Line , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Conserved Sequence/genetics , Cyclin-Dependent Kinase Inhibitor p15/genetics , Diabetes Mellitus, Type 2/genetics , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Gene Knockdown Techniques , Genome-Wide Association Study , Haplotypes/genetics , HeLa Cells , Humans , Interferon-alpha/genetics , Linkage Disequilibrium , Male , Polymorphism, Single Nucleotide/genetics , Protein Binding/drug effects , Purine-Nucleoside Phosphorylase/genetics , STAT1 Transcription Factor/biosynthesis , STAT1 Transcription Factor/deficiency , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , White People/geneticsABSTRACT
With the recent recognition of non-coding RNAs (ncRNAs) flanking many genes, a central issue is to obtain a full understanding of their potential roles in regulated gene transcription programmes, possibly through different mechanisms. Here we show that an RNA-binding protein, TLS (for translocated in liposarcoma), serves as a key transcriptional regulatory sensor of DNA damage signals that, on the basis of its allosteric modulation by RNA, specifically binds to and inhibits CREB-binding protein (CBP) and p300 histone acetyltransferase activities on a repressed gene target, cyclin D1 (CCND1) in human cell lines. Recruitment of TLS to the CCND1 promoter to cause gene-specific repression is directed by single-stranded, low-copy-number ncRNA transcripts tethered to the 5' regulatory regions of CCND1 that are induced in response to DNA damage signals. Our data suggest that signal-induced ncRNAs localized to regulatory regions of transcription units can act cooperatively as selective ligands, recruiting and modulating the activities of distinct classes of RNA-binding co-regulators in response to specific signals, providing an unexpected ncRNA/RNA-binding protein-based strategy to integrate transcriptional programmes.