Biology subjects

Dohmae, N.

Publications and source records attributed to Dohmae, N..

23 records · Page 2Linked to original sources

bioRxiv2020

Functional correlation of H3K9me2 and nuclear compartment formation

BackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation. ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

molecular biology↗

bioRxiv2020

Homeostatic regulation of STING by Golgi-to-ER membrane traffic

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi to the ER1,2. Mutation of the COPA gene, encoding one of the COP-I subunits (-COP), causes an immune dysregulatory disease (COPA syndrome)3. The molecular mechanism by which the impaired retrograde transport results in autoinflammation is not understood. Here we report that STING4, an innate immunity protein, is a cargo of the Golgi-to-ER membrane transport. In the presence of the disease-causative -COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signalling pathway. Surf45, a protein that circulates between the ER and the Golgi, binds STING and -COP, and mediates retrograde transport of STING to the ER. STING/Surf4/-COP complex is disrupted in the presence of the disease-causative -COP variant. Intriguingly, the STING ligand cGAMP also impairs the formation of STING/Surf4/-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by the Golgi-to-ER membrane traffic and provide insights into the pathogenesis of COPA syndrome.

cell biology↗

bioRxiv2019

Assessment of a western blot signal for the Bcnt/Cfdp1, a tentative component of Srcap chromatin remodeling complex; trial to overcome off-target problems

The BCNT (Bucentaur) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in fungi, fly, and chicken. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the Srcap (SNF2-Related CBP Activator Protein) chromatin remodeling complex, but little is known about its properties, partly because there are few suitable antibodies to detect the endogenous protein. We used multiple anti-Bcnt/Cfdp1 antibodies against unrelated immunogens derived from BCNT-C domain and mouse-specific N-terminal peptide. To assign western blot signals and evaluate these antibodies, we utilized a stem cell line from mutant embryos of mouse Bcnt/Cfdp1, whose mRNA expression levels were reduced to 75% of the parental cells. In western blotting of these mutant and parental cell extracts with the anti-Bcnt/Cfdp1 antibodies, mouse Bcnt/Cfdp1 was detected as a doublet of approximately 45 kDa. LC-MS/MS analysis of the corresponding doublet for the Flag-tagged mouse Bcnt/Cfdp1 constitutively expressed in T-REx 293 cell (a HEK293 derivative) exhibited that the upper band was much more phosphorylated than the lower band and that there was preferential Ser phosphorylation in the WESF motif in the BCNT-C domain. Western blot with these validated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development in mouse and rat, which is consistent with the expression of Bcnt/Cfdp1 mRNA. This article describes the evaluation of anti-Bcnt/Cfdp1 antibodies, including a scheme to prepare a potential negative control for western blot, and discusses immune-cross reactions with off-target proteins, particularly immunoreaction probabilities.

biochemistry↗

bioRxiv2018

Triazole ureas covalently bind to strigolactone receptors and regulate signaling

Strigolactones (SLs), a class of plant hormones with multiple functions, mediate plant-plant and plant-microorganism communications in the rhizosphere. In this study, we developed potent strigolactone antagonists, which covalently bind to the strigolactone receptor D14, by preparing an array of triazole urea compounds. Using yeast two-hybrid assays and rice tillering assays, we identified a triazole urea compound KK094 as a potent inhibitor of strigolactone receptors. The LC-MS/MS analysis and X-ray crystallography concluded that KK094 was hydrolyzed by D14, and that a reaction product of this degradation covalently binds to the Ser residue of the catalytic triad of D14. We also identified KK052 and KK073, whose effects on D14-D53/D14-SLR1 complex formation were opposite due to a trifluoromethyl group on its benzene ring. These results demonstrate that triazole urea compounds are potentially powerful tools for agricultural application and may be useful for the elucidation of the complicated mechanism underlying SL-perception.

plant biology↗

bioRxiv2018

Cryo-EM structure of the volume-regulated anion channel LRRC8

Maintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

molecular biology↗