Light‐Activation of DNA‐Methyltransferases

Wolffgramm, Jan; Buchmuller, Benjamin; Palei, Shubhendu; Muñoz‐López, Álvaro; Kanne, Julian; Janning, Petra; Schweiger, Michal R.; Summerer, Daniel

doi:10.1002/anie.202103945

Cited by 11 publications

(5 citation statements)

References 53 publications

(18 reference statements)

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“…We envisaged a replacement with 4,5‐dimethoxy‐2‐nitrobenzyl‐L‐serine 1 (Figure 1c) that can be incorporated into proteins in response to the amber codon (TAG) in mammalian cells via an engineered Escherichia coli leucyl‐tRNA‐synthetase (LRS)/tRNA Leu amber suppressor pair. [ 5a,9 ] Light‐irradiation of 1 allows effective, scarless decaging to the natural serine residue. Molecular modelling studies using GROMACS [ 10 ] based on an MBD1 NMR solution structure [ 8 ] suggested T27 and S45 as promising candidates (Figure 1a,b; T27 is replaced by a serine in MBD2‐3 and MeCP2).…”

Section: Resultsmentioning

confidence: 99%

Light‐Activatable MBD‐Readers of 5‐Methylcytosine Reveal Domain‐Dependent Chromatin Association Kinetics In Vivo

Lin,

Engelhard,

Söldner

et al. 2024

Advanced Science

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Abstract5‐Methylcytosine (5mC) is the central epigenetic mark of mammalian DNA, and plays fundamental roles in chromatin regulation. 5mC is dynamically read and translated into regulatory outputs by methyl‐CpG‐binding domain (MBD) proteins. These multidomain readers recognize 5mC via an MBD domain, and undergo additional domain‐dependent interactions with multiple additional chromatin components. However, studying this dynamic process is limited by a lack of methods to conditionally control the 5mC affinity of MBD readers in cells. Light‐control of MBD association to chromatin by genetically encoding a photocaged serine at the MBD‐DNA interface is reported. The authors study the association of MBD1 to mouse pericentromeres, dependent on its CxxC3 and transcriptional repressor domains (TRD) which interact with unmethylated CpG and heterochromatin‐associated complexes, respectively. Both domains significantly modulate association kinetics, arguing for a model in which the CxxC3 delays methylation responses of MBD1 by holding it at unmethylated loci, whereas the TRD promotes responses by aiding heterochromatin association is studied. Their approach offers otherwise inaccessible kinetic insights into the domain‐specific regulation of a central MBD reader, and sets the basis for further unravelling how the integration of MBDs into complex heterochromatin interaction networks control the kinetics of 5mC reading and translation into altered chromatin states.

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Section: Resultsmentioning

confidence: 99%

Light‐Activatable MBD‐Readers of 5‐Methylcytosine Reveal Domain‐Dependent Chromatin Association Kinetics In Vivo

Lin,

Engelhard,

Söldner

et al. 2024

Advanced Science

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“…Encouragingly, the genetic incorporation of photolabile UAAs to proteins has proven widely useful in the past decades, and photoswitchable proteins have also been engineered with azobenzene-based caging groups [ 147 ]. This unique opto-chemical tool for controlling proteins has been extensively employed in the study of numerous biological processes [ 148 , 149 , 150 ] including control of ion channels [ 146 , 151 , 152 , 153 ], kinase and phosphatase activities [ 154 , 155 , 156 ], gene expression [ 157 , 158 ], epigenetic regulation [ 159 , 160 ], DNA recombination [ 161 ], protein localization [ 162 , 163 ], protein self-organization [ 164 ], intein splicing [ 165 , 166 ], and O-linked-N-acetylglucosaminylation [ 167 ].…”

Section: An Overview Of Established Opto-chemical Toolsmentioning

confidence: 99%

The Development and Application of Opto-Chemical Tools in the Zebrafish

et al. 2022

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The zebrafish is one of the most widely adopted animal models in both basic and translational research. This popularity of the zebrafish results from several advantages such as a high degree of similarity to the human genome, the ease of genetic and chemical perturbations, external fertilization with high fecundity, transparent and fast-developing embryos, and relatively low cost-effective maintenance. In particular, body translucency is a unique feature of zebrafish that is not adequately obtained with other vertebrate organisms. The animal’s distinctive optical clarity and small size therefore make it a successful model for optical modulation and observation. Furthermore, the convenience of microinjection and high embryonic permeability readily allow for efficient delivery of large and small molecules into live animals. Finally, the numerous number of siblings obtained from a single pair of animals offers large replicates and improved statistical analysis of the results. In this review, we describe the development of opto-chemical tools based on various strategies that control biological activities with unprecedented spatiotemporal resolution. We also discuss the reported applications of these tools in zebrafish and highlight the current challenges and future possibilities of opto-chemical approaches, particularly at the single cell level.

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“…This has been applied to human KDAC8 to identify novel substrates (Lopez et al, 2017). Next to photocrosslinkers, other genetic code expansion systems allow the site-specific incorporation of fluorescent amino acids, some of which are even photocageable, such as o-nitrobenzoyl-lysine, to follow protein dynamics in cells enabling light control of cellular processes using live cell fluorescence microscopy (Gautier et al, 2010(Gautier et al, , 2011Lopez et al, 2017;Illes et al, 2021;Wolffgramm et al, 2021). Moreover, systems to genetically encode ubiquitin, phosphoserine and phosphothreonine were also developed that enables to study the impact of ubiquitination and phosphorylation on protein function (Chin, 2011(Chin, , 2017Park et al, 2011;Rogerson et al, 2015;de la Torre and Chin, 2021).…”

Section: Synthetic Biology: Genetically Encoding Acetyl-l-lysine In Proteins Allows To Unravel the Real Consequences Of Lysine Acetylatiomentioning

confidence: 99%

Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective

Lammers

2021

Front. Microbiol.

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Ac(et)ylation is a post-translational modification present in all domains of life. First identified in mammals in histones to regulate RNA synthesis, today it is known that is regulates fundamental cellular processes also in bacteria: transcription, translation, metabolism, cell motility. Ac(et)ylation can occur at the ε-amino group of lysine side chains or at the α-amino group of a protein. Furthermore small molecules such as polyamines and antibiotics can be acetylated and deacetylated enzymatically at amino groups. While much research focused on N-(ε)-ac(et)ylation of lysine side chains, much less is known about the occurrence, the regulation and the physiological roles on N-(α)-ac(et)ylation of protein amino termini in bacteria. Lysine ac(et)ylation was shown to affect protein function by various mechanisms ranging from quenching of the positive charge, increasing the lysine side chains’ size affecting the protein surface complementarity, increasing the hydrophobicity and by interfering with other post-translational modifications. While N-(ε)-lysine ac(et)ylation was shown to be reversible, dynamically regulated by lysine acetyltransferases and lysine deacetylases, for N-(α)-ac(et)ylation only N-terminal acetyltransferases were identified and so far no deacetylases were discovered neither in bacteria nor in mammals. To this end, N-terminal ac(et)ylation is regarded as being irreversible. Besides enzymatic ac(et)ylation, recent data showed that ac(et)ylation of lysine side chains and of the proteins N-termini can also occur non-enzymatically by the high-energy molecules acetyl-coenzyme A and acetyl-phosphate. Acetyl-phosphate is supposed to be the key molecule that drives non-enzymatic ac(et)ylation in bacteria. Non-enzymatic ac(et)ylation can occur site-specifically with both, the protein primary sequence and the three dimensional structure affecting its efficiency. Ac(et)ylation is tightly controlled by the cellular metabolic state as acetyltransferases use ac(et)yl-CoA as donor molecule for the ac(et)ylation and sirtuin deacetylases use NAD+ as co-substrate for the deac(et)ylation. Moreover, the accumulation of ac(et)yl-CoA and acetyl-phosphate is dependent on the cellular metabolic state. This constitutes a feedback control mechanism as activities of many metabolic enzymes were shown to be regulated by lysine ac(et)ylation. Our knowledge on lysine ac(et)ylation significantly increased in the last decade predominantly due to the huge methodological advances that were made in fields such as mass-spectrometry, structural biology and synthetic biology. This also includes the identification of additional acylations occurring on lysine side chains with supposedly different regulatory potential. This review highlights recent advances in the research field. Our knowledge on enzymatic regulation of lysine ac(et)ylation will be summarized with a special focus on structural and mechanistic characterization of the enzymes, the mechanisms underlying non-enzymatic/chemical ac(et)ylation are explained, recent technological progress in the field are presented and selected examples highlighting the important physiological roles of lysine ac(et)ylation are summarized.

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Light‐Activation of DNA‐Methyltransferases

Cited by 11 publications

References 53 publications

Light‐Activatable MBD‐Readers of 5‐Methylcytosine Reveal Domain‐Dependent Chromatin Association Kinetics In Vivo

Light‐Activatable MBD‐Readers of 5‐Methylcytosine Reveal Domain‐Dependent Chromatin Association Kinetics In Vivo

The Development and Application of Opto-Chemical Tools in the Zebrafish

Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective

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