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International Journal of Molecular... Jul 2023Ecdysone signaling in remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind...
Ecdysone signaling in remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. Here, we present experimental data on 11 various coregulators at ecdysone-responsive loci of S2 cells. We describe the regulatory elements where coregulators reside within these loci and assess changes in their binding levels following 20-hydroxyecdysone treatment. In the current study, we detected the presence of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. We observed minor changes in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. Our findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, our findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response.
Topics: Animals; Drosophila; Ecdysone; Drosophila Proteins; Ecdysterone; Receptors, Steroid; Transcriptional Activation; Drosophila melanogaster; Microtubule-Associated Proteins; Nuclear Proteins
PubMed: 37511602
DOI: 10.3390/ijms241411844 -
Proceedings of the National Academy of... Aug 2023Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic...
Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic RNA-binding sterile-α motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). Here, we resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 1.6 Å resolution, which revealed its composition of a homodimerization D subdomain and a subdomain with similarity to a pseudo-HEAT-repeat analogous topology (PHAT) domain. Furthermore, we show that Smaug directly interacts with the germline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its NTD. We determined the crystal structure of the NTD of Smaug in complex with a Smoothened α-helical peptide to 2.0 Å resolution. The peptide binds within a groove that is formed by both the D and PHAT subdomains. Structural modeling supported by experimental data suggested that an α-helix within the disordered region of Oskar binds to the NTD of Smaug in a mode similar to Smoothened. Together, our data uncover the NTD of Smaug as a peptide-binding domain.
Topics: Animals; Humans; Drosophila; Drosophila Proteins; Germ Cells; Hedgehog Proteins; RNA, Messenger; RNA-Binding Proteins; Repressor Proteins; Receptors, G-Protein-Coupled
PubMed: 37523566
DOI: 10.1073/pnas.2304385120 -
Aging Cell Oct 2023Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. Here, we report that adult-onset...
Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. Here, we report that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, we conducted a series of lifespan rescue experiments and the results indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, we showed that GLAD encoded a heterochromatin-associating protein that bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, our findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Longevity; Neuroglia
PubMed: 37594178
DOI: 10.1111/acel.13947 -
Developmental Cell Jul 2023Heart development begins with the formation of a tube as cardiac progenitors migrate from opposite sides of the embryo. Abnormal cardiac progenitor movements cause...
Heart development begins with the formation of a tube as cardiac progenitors migrate from opposite sides of the embryo. Abnormal cardiac progenitor movements cause congenital heart defects. However, the mechanisms of cell migration during early heart development remain poorly understood. Using quantitative microscopy, we found that in Drosophila embryos, cardiac progenitors (cardioblasts) migrated through a sequence of forward and backward steps. Cardioblast steps were associated with oscillatory non-muscle myosin II waves that induced periodic shape changes and were necessary for timely heart tube formation. Mathematical modeling predicted that forward cardioblast migration required a stiff boundary at the trailing edge. Consistent with this, we found a supracellular actin cable at the trailing edge of the cardioblasts that limited the amplitude of the backward steps, thus biasing the direction of cell movement. Our results indicate that periodic shape changes coupled with a polarized actin cable produce asymmetrical forces that promote cardioblast migration.
Topics: Animals; Drosophila; Drosophila Proteins; Actins; Heart; Myosins; Morphogenesis; Drosophila melanogaster
PubMed: 37295436
DOI: 10.1016/j.devcel.2023.05.005 -
PLoS Biology Jul 20231p32.3 microdeletion/duplication is implicated in many neurodevelopmental disorders-like phenotypes such as developmental delay, intellectual disability, autism,...
1p32.3 microdeletion/duplication is implicated in many neurodevelopmental disorders-like phenotypes such as developmental delay, intellectual disability, autism, macro/microcephaly, and dysmorphic features. The 1p32.3 chromosomal region harbors several genes critical for development; however, their validation and characterization remain inadequate. One such gene is the single-stranded DNA-binding protein 3 (SSBP3) and its Drosophila melanogaster ortholog is called sequence-specific single-stranded DNA-binding protein (Ssdp). Here, we investigated consequences of Ssdp manipulations on neurodevelopment, gene expression, physiological function, and autism-associated behaviors using Drosophila models. We found that SSBP3 and Ssdp are expressed in excitatory neurons in the brain. Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head. Ssdp manipulations also affected the neuropil brain volume and glial cell number in larvae and adult flies. Moreover, Ssdp overexpression led to differential changes in synaptic density in specific brain regions. We observed decreased levels of armadillo in the heads of Ssdp overexpressing flies, as well as a decrease in armadillo and wingless expression in the larval wing discs, implicating the involvement of the canonical Wnt signaling pathway in Ssdp functionality. RNA sequencing revealed perturbation of oxidative stress-related pathways in heads of Ssdp overexpressing flies. Furthermore, Ssdp overexpressing brains showed enhanced reactive oxygen species (ROS), altered neuronal mitochondrial morphology, and up-regulated fission and fusion genes. Flies with elevated levels of Ssdp exhibited heightened anxiety-like behavior, altered decisiveness, defective sensory perception and habituation, abnormal social interaction, and feeding defects, which were phenocopied in the pan-neuronal Ssdp knockdown flies, suggesting that Ssdp is dosage sensitive. Partial rescue of behavioral defects was observed upon normalization of Ssdp levels. Notably, Ssdp knockdown exclusively in adult flies did not produce behavioral and functional defects. Finally, we show that optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors. Collectively, our findings provide evidence that Ssdp, a dosage-sensitive gene in the 1p32.3 chromosomal region, is associated with various anatomical, physiological, and behavioral defects, which may be relevant to neurodevelopmental disorders like autism. Our study proposes SSBP3 as a critical gene in the 1p32.3 microdeletion/duplication genomic region and sheds light on the functional role of Ssdp in neurodevelopmental processes in Drosophila.
Topics: Animals; Humans; Armadillos; Autistic Disorder; DNA-Binding Proteins; Drosophila; Drosophila melanogaster; Drosophila Proteins; Transcription Factors
PubMed: 37486945
DOI: 10.1371/journal.pbio.3002210 -
RNA (New York, N.Y.) Aug 2023RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of...
RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of cells, we developed TRIBE, in which the catalytic domain of the RNA editing enzyme ADAR (ADARcd) is fused to an RBP. When the RBP binds to an mRNA, ADAR catalyzes A to G modifications in the target mRNA that can be easily identified in standard RNA sequencing. In STAMP, the concept is the same except the ADARcd is replaced by the RNA editing enzyme APOBEC. Here we compared TRIBE and STAMP side-by-side in human and cells. The goal is to learn the pros and cons of each method so that researchers can choose the method best suited to their RBP and system. In human cells, TRIBE and STAMP were performed using the RBP TDP-43. Although they both identified TDP-43 target mRNAs, combining the two methods more successfully identified high-confidence targets. In cells, RBP-APOBEC fusions generated only low numbers of editing sites, comparable to the level of control editing. This was true for two different RBPs, Hrp48 and Thor ( EIF4E-BP), indicating that STAMP does not work well in .
Topics: Animals; Humans; Drosophila; Drosophila Proteins; RNA, Messenger; RNA-Binding Proteins; DNA-Binding Proteins
PubMed: 37169395
DOI: 10.1261/rna.079608.123 -
Cell Reports Nov 2023Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between...
Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. Here, we introduce PromoterSwitch, a genetic platform we established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. We show that cancer-driving genetic alterations representing common colon tumor genome landscapes disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. We also show that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy.
Topics: Animals; Drosophila; Signal Transduction; Intestines; Cell Differentiation; Enteroendocrine Cells; Cell Lineage; Drosophila Proteins; Carcinogenesis; Neoplasms
PubMed: 37924517
DOI: 10.1016/j.celrep.2023.113370 -
Cell Reports Nov 2023Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming...
Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming studies showing that the unfolded protein response (UPR) is dysregulated in various tumors, it remains debatable whether the UPR restrains or promotes tumorigenesis. Here, using Drosophila eye epithelium as a model, we uncover a surprising decisive role of the Ire1 branch of the UPR in regulating cell polarity gene scribble (scrib) loss-induced TSCC. Both mutation and hyperactivation of Ire1 accelerate elimination of scrib clones via inducing apoptosis and autophagy, respectively. Unexpectedly, relative Ire1 activity is also crucial for determining loser cell fate, as dysregulating Ire1 signaling in the surrounding healthy cells reversed the "loser" status of scrib clones by decreasing their apoptosis. Furthermore, we show that Ire1 is required for cell competition in mammalian cells. Together, these findings provide molecular insights into scrib-mediated TSCC and highlight Ire1 as a key determinant of loser cell fate.
Topics: Animals; Cell Competition; Drosophila; Drosophila Proteins; Mammals; Neoplasms; Signal Transduction; Unfolded Protein Response
PubMed: 37924514
DOI: 10.1016/j.celrep.2023.113303 -
Proceedings of the National Academy of... Aug 2023Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. Here, we describe a striking organization of mitochondria into a...
Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. Here, we describe a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeiotic spermatocytes, which we term "mitoball". Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. We observed similar premeiotic mitochondrial clusters in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, we identified that Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. We suggest that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development.
Topics: Animals; Male; Drosophila melanogaster; Drosophila Proteins; Mitochondria; Semen; Spermatogenesis; Nerve Tissue Proteins
PubMed: 37579146
DOI: 10.1073/pnas.2306073120 -
Genesis (New York, N.Y. : 2000) Feb 2024Hox genes encode transcription factors that play an important role in establishing the basic body plan of animals. In Drosophila, Antennapedia is one of the five genes... (Review)
Review
Hox genes encode transcription factors that play an important role in establishing the basic body plan of animals. In Drosophila, Antennapedia is one of the five genes that make up the Antennapedia complex (ANT-C). Antennapedia determines the identity of the second thoracic segment, known as the mesothorax. Misexpression of Antennapedia at different developmental stages changes the identity of the mesothorax, including the muscles, nervous system, and cuticle. In Drosophila, Antennapedia has two distinct promoters highly regulated throughout development by several transcription factors. Antennapedia proteins are found with other transcription factors in different ANTENNAPEDIA transcriptional complexes to regulate multiple subsets of target genes. In this review, we describe the different mechanisms that regulate the expression and function of Antennapedia and the role of this Hox gene in the development of Drosophila.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Gene Expression Regulation, Developmental; Homeodomain Proteins; Transcription Factors
PubMed: 37830148
DOI: 10.1002/dvg.23561