The tissue- and developmental stage-specific involvement of autophagy genes in aggrephagy.
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ABSTRACT:
SUBMITTER: Zheng H
PROVIDER: S-EPMC7138198 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil-DNA glycosylase and dUTPase. Lack of the major uracil-DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200-2,000 uracil/million bases, quantified using a novel real-time PCR-based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil-DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil-DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil-DNA in this evolutionary clade.
| S-EPMC3369950 | biostudies-literature
Project description:BackgroundMicroRNAs (miRNAs) regulate the expression of target genes by mediating gene silencing in both plants and animals. The miRNA targets have been extensively investigated in Arabidopsis and rice using computational prediction, experimental validation by overexpression in transgenic plants, and by degradome or PARE (parallel analysis of RNA ends) sequencing. However, miRNA targets mostly remain unknown in soybean (Glycine max). More specifically miRNA mediated gene regulation at different seed developmental stages in soybean is largely unexplored. In order to dissect miRNA guided gene regulation in soybean developing seeds, we performed a transcriptome-wide experimental method using degradome sequencing to directly detect cleaved miRNA targets.ResultsIn this study, degradome libraries were separately prepared from immature soybean cotyledons representing three stages of development and from seed coats of two stages. Sequencing and analysis of 10 to 40 million reads from each library resulted in identification of 183 different targets for 53 known soybean miRNAs. Among these, some were found only in the cotyledons representing cleavage by 25 miRNAs and others were found only in the seed coats reflecting cleavage by 12 miRNAs. A large number of targets for 16 miRNAs families were identified in both tissues irrespective of the stage. Interestingly, we identified more miRNA targets in the desiccating cotyledons of late seed maturation than in immature seed. We validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5' rapid amplification of cDNA ends (RLM-5'RACE). Gene Ontology (GO) analysis indicated the involvement of miRNA target genes in various cellular processes during seed development.ConclusionsThe miRNA targets in both the cotyledons and seed coats of several stages of soybean seed development have been elucidated by experimental evidence from comprehensive, high throughput sequencing of the enriched fragments resulting from miRNA-guided cleavage of messenger RNAs. Nearly 50% of the miRNA targets were transcription factors in pathways that are likely important in setting or maintaining the developmental program leading to high quality soybean seeds that are one of the dominant sources of protein and oil in world markets.
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Project description:BackgroundRecent years have seen an explosion in plant genomics, as the difficulties inherent in sequencing and functionally analyzing these biologically and economically significant organisms have been overcome. Arabidopsis thaliana, a versatile model organism, represents an opportunity to evaluate the predictive power of biological network inference for plant functional genomics.ResultsHere, we provide a compendium of functional relationship networks for Arabidopsis thaliana leveraging data integration based on over 60 microarray, physical and genetic interaction, and literature curation datasets. These include tissue, biological process, and development stage specific networks, each predicting relationships specific to an individual biological context. These biological networks enable the rapid investigation of uncharacterized genes in specific tissues and developmental stages of interest and summarize a very large collection of A. thaliana data for biological examination. We found validation in the literature for many of our predicted networks, including those involved in disease resistance, root hair patterning, and auxin homeostasis.ConclusionsThese context-specific networks demonstrate that highly specific biological hypotheses can be generated for a diversity of individual processes, developmental stages, and plant tissues in A. thaliana. All predicted functional networks are available online at http://function.princeton.edu/arathGraphle.
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Project description:The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease.
| S-EPMC6761021 | biostudies-literature
Project description:BACKGROUND:Identifying DNA sequences (enhancers) that direct the precise spatial and temporal expression of developmental control genes remains a significant challenge in the annotation of vertebrate genomes. Locating these sequences, which in many cases lie at a great distance from the transcription start site, has been a major obstacle in deciphering gene regulation. Coupling of comparative genomics with functional validation to locate such regulatory elements has been a successful method in locating many such regulatory elements. But most of these studies looked either at a single gene only or the whole genome without focusing on any particular process. The pressing need is to integrate the tools of comparative genomics with knowledge of developmental biology to validate enhancers for developmental transcription factors in greater detail RESULTS:Our results show that near four different genes (nkx3.2, pax9, otx1b and foxa2) in zebrafish, only 20-30% of highly conserved DNA sequences can act as developmental enhancers irrespective of the tissue the gene expresses in. We find that some genes also have multiple conserved enhancers expressing in the same tissue at the same or different time points in development. We also located non-conserved enhancers for two of the genes (pax9 and otx1b). Our modified Bacterial artificial chromosome (BACs) studies for these 4 genes revealed that many of these enhancers work in a synergistic fashion, which cannot be captured by individual DNA constructs and are not conserved at the sequence level. Our detailed biochemical and transgenic analysis revealed Foxa1 binds to the otx1b non-conserved enhancer to direct its activity in forebrain and otic vesicle of zebrafish at 24 hpf. CONCLUSION:Our results clearly indicate that high level of functional conservation of genes is not necessarily associated with sequence conservation of its regulatory elements. Moreover certain non conserved DNA elements might have role in gene regulation. The need is to bring together multiple approaches to bear upon individual genes to decipher all its regulatory elements.
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Project description:Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of "autophagy profiles" revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.Abbreviations: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-β: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.
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Project description:The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.
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Project description: Not available
| S-EPMC10794840 | biostudies-literature
Project description:Isolation of individual cells ensures detailed analysis of human embryos and promotes our understanding of molecular mechanisms driving embryo development and cell specification. Here, we present a protocol for the processing of human embryos for single-cell analysis. We describe steps for growing embryos and individualizing cells from the polar and the mural parts of trophectoderm at the blastocyst stage using laser dissection. We then detail embryo dissociation followed by steps to pick, wash, and dispense cells in plates.
| S-EPMC10300299 | biostudies-literature
Project description:Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.
| S-EPMC2814043 | biostudies-literature