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Cho, U.-S.

Publications and source records attributed to Cho, U.-S..

7 recordsLinked to original sources

A neuron-specific microexon ablates the novel DNA-binding function of a histone H3K4me0 reader PHF21A

How cell-type-specific chromatin landscapes emerge and progress during metazoan ontogenesis remains an important question. Transcription factors are expressed in a cell-type-specific manner and recruit chromatin-regulatory machinery to specific genomic loci. In contrast, chromatin-regulatory proteins are expressed broadly and are assumed to exert the same intrinsic function across cell types. However, human genetics studies have revealed an unexpected vulnerability of neurodevelopment to chromatin factor mutations with unknown mechanisms. Here, we report that 14 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Of the 14 chromatin regulators, two are integral components of a histone H3K4 demethylase complex; the catalytic subunit LSD1 and an H3K4me0-reader protein PHF21A adopt neuron-specific forms. We found that canonical PHF21A (PHF21A-c) binds to DNA by AT-hook motif, and the neuronal counterpart PHF21A-n lacks this DNA-binding function yet maintains H3K4me0 recognition intact. In-vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes identified the neuronal complex as a hypomorphic H3K4 demethylating machinery with reduced nucleosome engagement. Furthermore, an autism-associated PHF21A missense mutation, 1285 G>A, at the last nucleotide of the common exon immediately upstream of the neuronal microexon led to impaired splicing of PHF21A-n. Thus, ubiquitous chromatin regulatory complexes exert unique intrinsic functions in neurons via alternative splicing of their subunits and potentially contribute to faithful human brain development.

molecular biology↗

Structural Basis for Mis18 Complex Assembly: Implications for Centromere Maintenance

The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18, Mis18y and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18, 2 Mis18y and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18 can associate with centromeres and deposit CENP-A independently of Mis18y, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

cell biology↗

Atmospheric plasma jet device for versatile electron microscope grid treatment

Atmospheric pressure plasmas have been widely applied in surface modification and biomedical treatment due to its ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used in eliminating the surface contaminants as well as generating the hydrophilic surface to embed the specimen on grids. Plasma treatment is a prerequisite for negative stain and quantifoil grids, which are coated with hydrophobic carbon on the grid surface. Here we introduce a non-thermal atmospheric plasma jet system as an alternative new tool for surface treatment. Unlike the conventional glow discharger, we found that the plasma jet system successfully cleans the grid surface and introduces hydrophilicity on grids in the ambient environment without introducing a vacuum. Therefore, we anticipate the plasma jet system will be beneficial in many aspects, such as cost-effective, convenient, versatile, and potential applications in surface modification for both negative stain and cryo-EM grid treatment.

biochemistry↗

MLL1 Methyltransferase Activity is Regulated by Distinct Nucleosome Binding Modes

Here we solve the single particle cryoEM structure for the MLL1 complex with nucleosome core particle (NCP) carrying histone H3 lysine 4 to methionine mutation. The MLL1 complex displays significant rotational dynamics on the NCP, a feature distinct from the yeast SET1 complex. We identified two major binding modes of the MLL1 complex on the NCP. Both binding modes anchor on the NCP through ASH2L, but they differ drastically with regard to where the MLL1 SET domain and RbBP5 bind. We show that one of the binding modes is catalytically inactive since disrupting interactions unique to this binding mode does not affect overall MLL1 activity in an NCP-specific manner. Interestingly, the inactive binding mode is in a configuration similar to that of the ySET1- NCP complex, which is intrinsically inactive on an unmodified NCP. The high rotational dynamics of the MLL1 complex as well as distinction between MLL and yeast SET1 complexes may reflect the necessity for loci-specific regulation of H3K4 methylation states in higher eukaryotes.

biochemistry↗

Dismantling and rebuilding the trisulfide cofactor demonstrates its essential role in human sulfide quinone oxidoreductase

Sulfide quinone oxidoreductase (SQR) catalyzes the first step in sulfide clearance, coupling H2S oxidation to coenzyme Q reduction. Recent structures of human SQR revealed a sulfur atom bridging the SQR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic and computational modeling approaches. Cyanolysis of SQR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. Cyanolysis leads to reversible loss of SQR activity, which is restored in the presence of sulfide. We captured a crystallographic intermediate in SQR that provides clues as to how the oxidized state of the cysteines is preserved. Computational modeling and MD simulations revealed an ~105-fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.

biochemistry↗

MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions1-3. The maximum catalytic activity of hydroxylase (MMOH) is achieved via interplay of its regulatory protein (MMOB) and reductase4-6. An additional auxiliary protein, MMOD, is believed to function as an inhibitor of the catalytic activity of MMOH; however, the mechanism of its action remains unknown7,8. Herein, we report the crystal structure of MMOH-MMOD complex from Methylosinus sporium strain 5 (2.6 [A]), which illustrates that two molecules of MMOD associate symmetrically with the canyon region of MMOH in a manner similar to MMOB, indicating that MMOD competes with MMOB for MMOH recognition. Further, MMOD binding disrupts the geometry of the di-iron centre and opens the substrate access channel. Notably, the electron density of 1,6-hexanediol at the substrate access channel mimics products of sMMO in hydrocarbon oxidation. The crystal structure of MMOH-MMOD unravels the inhibitory mechanism by which MMOD suppresses the MMOH catalytic activity, and reveals how hydrocarbon substrates/products access to the di-iron centre.

biochemistry↗

Structural insights into the architecture of human Importin4_histone H3/H4_Asf1a complex and its histone H3 tail binding

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The XL-MS data showed that the C-terminal region of Importin4 interacts extensively with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic path in Importin4 accommodates the histone H3 tail and that histone H3 Lys14 is the primary residue interacting with Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.

biochemistry↗