Probing Chromatin-modifying Enzymes with Chemical Tools

Abstract

2 Glossary section/Key words:Epigenetics: Inheritance of gene expression profiles independent of changes in DNA sequence or content.Histones: Small, basic proteins that package eukaryotic DNA into chromatin.Nucleosomes: Nucleosome core particles consist of an octamer of histone proteins (2x H2A, H2B, H3, H4) around which 147 bp of DNA are wrapped. Addition of linker DNA of various length and linker histones (H1) makes up the nucleosome as the basic, repeating unit of chromatin. DNA-Methylation:Enzymatic and none-enzymatic modification of DNA bases with methyl-groups.The methylation of C5 of cytosine is most relevant for gene regulation.Histone modifications: Chemical, posttranslational modifications of specific amino acids of histone proteins. The most abundant modifications are acetylation and methylation of the ε-amino group of lysines, methylation of the guanidino group of arginines and phosphorylation of hydroxyl groups in serines and threonines.Chemical probes: Synthetic molecules designed to interact with a protein of interest in order to study its function and activity.Cosubstrate probes: Chemical probes that are derived from the cellular small molecules that donate modifying groups to DNA and histones such as Adenosine triphosphate (ATP), Acetyl coenzyme A (acetyl-CoA) and S-adenosyl methionine (SAM).Substrate profiling: Proteomic approach to define the pool of target proteins of specific modifying enzymes using cosubstrate probes and mass spectrometry. 3 AbstractChromatin is the universal template of genetic information of all eukaryotic organisms. Chemical modifications of the DNA-packaging histone proteins and the DNA bases are crucial signaling events in directing the use and readout of eukaryotic genomes. The enzymes that install and remove these chromatin modifications as well as the proteins that bind these marks govern information that goes beyond the sequence of DNA. Therefore, these so called epigenetic regulators are intensively studied and represent promising drug targets in modern medicine. We summarize and discuss recent advances in the field of chemical biology that have provided chromatin research with sophisticated tools for investigating the composition, activity and target sites of chromatin modifying enzymes and reader proteins. 4In all eukaryotic cells chromatin, the complex of DNA and histone proteins regulates the functional state of the genome by altering condensation states and active recruitment of regulatory factors. The basic structural unit of chromatin is the nucleosome core particle. It consists of a protein core formed by two copies each of the histone proteins H2A, H2B, H3 and H4 with 147 base pairs of DNA wrapped around.1 Nucleosome core particles are connected by linker DNA, which can bind linker histones thereby giving rise to the fundamental repeating unit of chromatin, the nucleosome. Control of different chromatin states is mediated by a multitude of posttranslational modifications (PTMs) of the histone proteins, including methylation, acetylation and phosphorylatio...

Theoretical Investigation of the Binding of Nucleobases to Cucurbiturils by Dispersion Corrected DFT Approaches

“…The presence of the array of noncovalent interactions which include electrostatic, hydrogen bonding, and hydrophobic interactions makes the nucleobases responsible for the storage, expression, and transmission of genetic information . The presence of such interaction between the nucleobases leads to the development of functionalized nanomaterials with potential application in molecular therapeutics, in biomimetics, as 3D coordination polymers, and in electronics. , …”

The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

“…This can be complemented by autoradiography assays using radioactive substrates, which excel in sensitivity, and are employed to study, e.g., AMPylation (Neunuebel et al, 2011 ; Tan and Luo, 2011 ) or glycosyltransferase effectors (Jank et al, 2012 ). Non-radioactive chemical substrate analogs (CSAs), which can be functionalized to visualize and isolate modified proteins, were developed for several PTMs (Grammel et al, 2011 ; Lu et al, 2012 ; Fischle and Schwarzer, 2016 ). CSAs can also reveal PTMs on effectors, as demonstrated for the post-translational lipidation of effectors (Figure 2C.3 ) (Ivanov et al, 2010 ; Lin et al, 2015 ; Schroeder et al, 2015 ).…”

“…ABPs are typically small molecules that irreversibly react with a specific enzyme class and functionalized to allow purification of modified enzymes for MS analysis. ABPs are available for many enzymes including the ubiquitin-conjugation and chromatin-modifying machineries (Willems et al, 2014 ; Fischle and Schwarzer, 2016 ; Hewings et al, 2017 ). ABPs will help to disentangle the redundancy problem, probe for eukaryotic-like enzymes and assign functions in new Legionella isolates.…”

Visible‐Light Removable Photocaging Groups Accepted by MjMAT Variant: Structural Basis and Compatibility with DNA and RNA Methyltransferases

“…Histones are methylated on lysines and arginines, yielding different methylation states [1c] . In nucleic acids, DNA methyltransferases catalyze the installation of a methyl group at the N 4 or C5 position of cytosines and the N 6 position of adenines [2] . RNA methyltransferases act on many different positions in tRNAs and rRNAs [3] .…”

“…AdoMet analogues and AdoMet‐based inhibitors have been used as tools to study the mechanism and function of MTases [10] . It was shown that M.TaqI, a prokaryotic DNA MTase targeting the N 6 of adenine in the recognition sequence 5′‐TCGA*‐3′ [11] accepts a number of AdoMet analogues that are extended at the nucleobase, the sulfonium center or both [2,9a,b,12] . In particular, the AdoMet analogues with extended carbon chains (e. g. alkyl, alkenyl and alkynyl groups) replacing the methyl group at the sulfonium center proved valuable for labeling and isolation of modified MTase target sites [9a,d,g,13] .…”