ABSTRACT
A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and ß-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr's gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.
Subject(s)
Chromatin , Proteome , Acylation , Chromosome Mapping , Histones , Cell SurvivalABSTRACT
The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome-wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX-MS) and RNA sequencing (PhotoCAX-seq) for the study of the composition and dynamics of protein-RNA interactions. By integrating the blue light-triggered photocatalyst with a dual-functional RNA-protein crosslinker (RP-linker) and the phase separation-based enrichment strategy, PhotoCAX-MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS-stimulation, as well as the identification of RBPs interacting directly with the 5' untranslated regions of SARS-CoV-2 RNA.
Subject(s)
COVID-19 , RNA-Binding Proteins , Cell Survival , HEK293 Cells , Humans , RNA, Viral , RNA-Binding Proteins/metabolism , SARS-CoV-2ABSTRACT
Posttranslational modifications (PTMs) of lysine are crucial histone marks that regulate diverse biological processes. The functional roles and regulation mechanism of many newly identified lysine PTMs, however, remain yet to be understood. Here we report a photoaffinity crotonyl lysine (Kcr) analogue that can be genetically and site-specifically incorporated into histone proteins. This, in conjunction with the genetically encoded photo-lysine as a "control probe", enables the capture and identification of enzymatic machinery and/or effector proteins for histone lysine crotonylation.
Subject(s)
Histones/chemistry , Histones/genetics , Lysine/chemistry , Photoaffinity Labels/chemistry , Genetic Code , Histones/metabolism , Lysine/analogs & derivatives , Lysine/metabolism , Molecular Conformation , Photoaffinity Labels/metabolismABSTRACT
A series of novel indanone derivatives was designed, synthesised and evaluated as potential agents for Alzheimer's disease. Among them, compound 6a, with a piperidine group linked to indone by a two-carbon spacer, exhibited the most potent inhibitor activity, with an IC(50) of 0.0018 µM for AChE; the inhibitory activity of this compound was 14-fold more potent than that of donepezil. Furthermore, these compounds also exhibited good metal-chelating ability.
Subject(s)
Acetylcholinesterase/chemistry , Chelating Agents/chemical synthesis , Cholinesterase Inhibitors/chemical synthesis , Drug Design , Indans/chemical synthesis , Metals/chemistry , Pyridines/chemical synthesis , Acetylcholinesterase/metabolism , Alzheimer Disease/drug therapy , Chelating Agents/chemistry , Chelating Agents/therapeutic use , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/therapeutic use , Humans , Indans/chemistry , Indans/therapeutic use , Kinetics , Pyridines/chemistry , Pyridines/therapeutic use , Structure-Activity RelationshipABSTRACT
RNA polymerase II (Pol II) apparatuses are compartmentalized into transcriptional clusters. Whether protein factors control these clusters remains unknown. In this study, we find that the ATPase-associated with diverse cellular activities (AAA + ) ATPase RUVBL2 co-occupies promoters with Pol II and various transcription factors. RUVBL2 interacts with unphosphorylated Pol II in chromatin to promote RPB1 carboxy-terminal domain (CTD) clustering and transcription initiation. Rapid depletion of RUVBL2 leads to a decrease in the number of Pol II clusters and inhibits nascent RNA synthesis, and tethering RUVBL2 to an active promoter enhances Pol II clustering at the promoter. We also identify target genes that are directly linked to the RUVBL2-Pol II axis. Many of these genes are hallmarks of cancers and encode proteins with diverse cellular functions. Our results demonstrate an emerging activity for RUVBL2 in regulating Pol II cluster formation in the nucleus.
Subject(s)
RNA Polymerase II , Transcription Factors , Adenosine Triphosphatases/metabolism , Chromatin/genetics , Cluster Analysis , RNA , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Berberine derivatives with substituted amino groups linked at the 9-position using different carbon spacers were designed, synthesized, and biologically evaluated as inhibitors of acetylcholinesterase. Compound 10b, with a cyclohexylamino group linked to berberine by a three carbon spacer, gave the most potent inhibitor activity with an IC(50) of 0.020 µM for AChE. Kinetic studies revealed mixed inhibition of AChE, and molecular modeling simulations of the AChE-inhibitor complex confirmed that compounds bound to both the catalytic active site and the peripheral anionic site.