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Genes & Development Jun 2024Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by...
Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of , , and mutant We found that the gene expression profiles of and larvae are markedly different than larvae despite similar reductions in H4K20me1. mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of , indicating that the developmental defects of animals are largely due to H4K20me1-independent effects on gene expression. Furthermore, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in and mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin and therefore that H4K20me1 does not play a large role in gene expression.
Topics: Animals; Drosophila melanogaster; Histones; Drosophila Proteins; Methylation; Lysine; Histone-Lysine N-Methyltransferase; Gene Expression Regulation, Developmental; Mutation; Transcriptome; Larva
PubMed: 38866557
DOI: 10.1101/gad.351698.124 -
Genes & Development Jun 2024Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in involves changes in...
Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.
Topics: Animals; Oogenesis; Drosophila Proteins; Cell Differentiation; Nuclear Pore; Genome, Insect; Gene Expression Regulation, Developmental; Female; Drosophila melanogaster; Oocytes; Transcription Factors; Drosophila; Nuclear Pore Complex Proteins
PubMed: 38866556
DOI: 10.1101/gad.351402.123 -
Genes & Development Jun 2024The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the...
The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in and demonstrates the importance of gene repression at the nuclear periphery for cell fate.
Topics: Animals; Drosophila Proteins; Chromatin; Cell Nucleus; Female; Cell Differentiation; Drosophila melanogaster; Stem Cells; Gene Expression Regulation, Developmental; Drosophila; Germ Cells
PubMed: 38866555
DOI: 10.1101/gad.351424.123 -
Genes & Development Jun 2024Gene transcription is intimately linked to chromatin state and histone modifications. However, the enzymes mediating these post-translational modifications have many... (Review)
Review
Gene transcription is intimately linked to chromatin state and histone modifications. However, the enzymes mediating these post-translational modifications have many additional, nonhistone substrates, making it difficult to ascribe the most relevant modification. In this issue of , Crain and colleagues (doi:10.1101/gad.351698.124) have combined a powerful histone replacement system with mutational analysis of a chromatin regulator and a chromatin reader in Importantly, they discovered that genes controlled by the histone 4 lysine 20 (H4K20) methyltransferase Set8 and the protein recognizing H4K20 monomethylation, L(3)mbt, differ substantially from those affected by mutation of H4K20 itself. This demonstrates that H4K20 is not the key substrate for Set8 but that methylation of other, unidentified proteins mediates its effects on transcription.
Topics: Animals; Drosophila melanogaster; Chromatin; Histones; Drosophila Proteins; Histone-Lysine N-Methyltransferase; Methylation; Protein Processing, Post-Translational
PubMed: 38866554
DOI: 10.1101/gad.351969.124 -
Cold Spring Harbor Protocols Jun 2024In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized....
In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in the NMJ.
PubMed: 38866544
DOI: 10.1101/pdb.prot108502 -
Cold Spring Harbor Protocols Jun 2024Determining the precise localization of interacting proteins provides fundamental insight into their putative function. Classically, immunolabeling of endogenous...
Determining the precise localization of interacting proteins provides fundamental insight into their putative function. Classically, immunolabeling of endogenous proteins or generating tagged versions of proteins has been used to localize interacting proteins. However, in many cases, the interacting partner of a protein of interest is unknown. For cell surface proteins, it is possible to determine the localization of interacting proteins if one of the binding partners is known. This approach is based on generating purified, recombinant, tagged extracellular domains (ECDs) of a protein of interest, and incubating tissue to allow the recombinant protein to bind to its interacting partner(s). In this protocol, we detail the cloning of secreted, tagged ECDs from cell surface proteins, transfection of cloned plasmids into S2 cells, collection of secreted domains, concentration of the cell culture medium to enrich for the ECDs, and labeling of tissue with these ECDs.
PubMed: 38866541
DOI: 10.1101/pdb.prot108501 -
American Journal of Human Genetics Jun 2024Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1,...
Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, we have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. Our findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Our investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health.
PubMed: 38866022
DOI: 10.1016/j.ajhg.2024.05.016 -
Molecular Biology and Evolution Jun 2024Maintaining genome integrity is vital for organismal survival and reproduction. Essential, broadly conserved DNA repair pathways actively preserve genome integrity....
Maintaining genome integrity is vital for organismal survival and reproduction. Essential, broadly conserved DNA repair pathways actively preserve genome integrity. However, many DNA repair proteins evolve adaptively. Ecological forces like UV exposure are classically cited drivers of DNA repair evolution. Intrinsic forces like repetitive DNA, which also imperil genome integrity, have received less attention. We recently reported that a Drosophila melanogaster-specific DNA satellite array triggered species-specific, adaptive evolution of a DNA repair protein called Spartan/MH. The Spartan family of proteases cleave hazardous, covalent crosslinks that form between DNA and proteins ("DNA-protein crosslink repair"). Appreciating that DNA satellites are both ubiquitous and universally fast-evolving, we hypothesized that satellite DNA turnover spurs adaptive evolution of DNA-protein crosslink repair beyond a single gene and beyond the D. melanogaster lineage. This hypothesis predicts pervasive Spartan gene family diversification across Drosophila species. To study the evolutionary history of the Drosophila Spartan gene family, we conducted population genetic, molecular evolution, phylogenomic, and tissue-specific expression analyses. We uncovered widespread signals of positive selection across multiple Spartan family genes and across multiple evolutionary timescales. We also detected recurrent Spartan family gene duplication, divergence, and gene loss. Finally, we found that ovary-enriched parent genes consistently birthed functionally diverged, testis-enriched daughter genes. To account for Spartan family diversification, we introduce a novel mechanistic model of antagonistic coevolution that links DNA satellite evolution and adaptive regulation of Spartan protease activity. This framework promises to accelerate our understanding of how DNA repeats drive recurrent evolutionary innovation to preserve genome integrity.
Topics: Animals; Evolution, Molecular; Gene Duplication; DNA Repair; Drosophila Proteins; Phylogeny; Drosophila melanogaster; Drosophila; Multigene Family; Selection, Genetic; DNA, Satellite
PubMed: 38865490
DOI: 10.1093/molbev/msae113 -
Molecular Biology of the Cell Jun 2024The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein...
The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and co-orientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that the SPC105R C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for lateral microtubule attachments and bi-orientation of homologs, which are critical for accurate chromosome segregation in meiosis I.
PubMed: 38865189
DOI: 10.1091/mbc.E24-02-0067 -
Journal of Agricultural and Food... Jun 2024The cotton aphid, , is a polyphagous pest that stunts host plant growth via direct feeding or transmitting plant virus. Due to the long-term application of insecticides,...
The cotton aphid, , is a polyphagous pest that stunts host plant growth via direct feeding or transmitting plant virus. Due to the long-term application of insecticides, has developed different levels of resistance to numerous insecticides. We found that five field populations had evolved multiple resistances to neonicotinoids. To explore the resistance mechanism mediated by uridine diphosphate glycosyltransferases (UGTs), two upregulated UGT genes in these five strains, and , were selected for functional analysis of their roles in neonicotinoid detoxification. Transgenic bioassay results indicated that compared with the control lines, the and overexpression lines were more tolerant to thiamethoxam, imidacloprid, and dinotefuran. Knockdown of and significantly increased sensitivity to thiamethoxam, imidacloprid, and dinotefuran. Molecular docking analysis demonstrated that these neonicotinoids could bind to the active pockets of UGT350C3 and UGT344L7. This study provides functional evidence of neonicotinoid detoxification mediated by UGTs and will facilitate further work to identify strategies for preventing the development of neonicotinoid resistance in insects.
Topics: Animals; Aphids; Neonicotinoids; Insecticides; Insecticide Resistance; Glycosyltransferases; Nitro Compounds; Molecular Docking Simulation; Insect Proteins; Thiamethoxam; Drosophila; Guanidines
PubMed: 38864686
DOI: 10.1021/acs.jafc.4c02682