Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16INK4A , when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate chromatin structure and gene activity in eukaryotic organisms. Methylated DNA sequences are generally associated with long term transcriptional silencing through the recruitment of repressor complexes, including methyl-binding proteins, histone deacetylases, and chromatin remodeling machinery (1, 2). Likewise, specific histone methylation states can recruit multivalent adaptor proteins, which lead to chromatin condensation, further inhibiting gene expression. Accumulating evidence shows that these two methylation systems act cooperatively to establish the epigenetic state of the cell (3-5); however, the mechanisms of this cooperation remain vague.During replication, CpG methylation patterns are maintained in mammals by the DNA methyltransferase 1 with hemimethylated CpG dinucleotides serving as a substrate. This enzyme is aided by UHRF1 (ubiquitin-like, PHD and RING finger containing 1, also known as ICBP90 in humans and NP95 in mouse), which interacts with DNA methyltransferase 1 and specifically recognizes hemimethylated CpG dinucleotides through its SET-and RING-associated domain (SRA) 4 (6, 7). 4 The abbreviations used are: SRA, SET-and RING-associated domain; UHRF1, ubiquitin-like, PHD and RING finger containing 1; mUHRF1, murine UHRF1; TTD, tandem tudor domain; TTD N , N-terminal tudor subdomain; TTD C , C-terminal tudor subdomain; NMR, nuclear magnetic resonance; RDC, residual dipolar coupli...
Knowledge of the structure and dynamics of RNA molecules is critical to understand their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be significantly enhanced by methods that enable incorporation of modified or labeled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. We have developed a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labeling. We demonstrate its utility by successfully preparing various isotope- or fluorescently-labeled versions of the 71-nucleotide aptamer domain of an adenine riboswitch1 for nuclear magnetic resonance (NMR) spectroscopy or single molecule Förster resonance-energy transfer (smFRET), respectively. Those RNAs include molecules that were selectively isotope-labeled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently-labeled in and near kissing loops. These selectively labeled RNAs have the same fold as those transcribed using conventional methods, but greatly simplified the interpretation of NMR spectra. The single-position isotope-labeled and fluorescently-labeled RNA samples revealed multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labeling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection2 and disease diagnostics3,4.
The three-dimensional solution structure of the HIV-I protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV proteasehnhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5", respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang C H et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A.As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.