Browsing by Author "Choudalakis, Michel"

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Open Access
Biochemical investigations of multivalent chromatin reading domains
(2024) Choudalakis, Michel; Jeltsch, Albert (Prof. Dr.)
In eukaryotes, the negatively charged nuclear DNA wraps around cationic histone proteins to form nucleosomes and compact the genetic information. Histones carry several post-translational modifications (PTMs) that appear in combinatorial patterns. These marks are interpreted by non-covalent interactions with proteins containing histone modification interacting domains (HiMIDs), also known as “reader” domains. Thirty years ago, it was proposed that the histone marks act as signals in the regulation of transcription and other chromatin functions. With time, this concept has been refined to suggest that combinatorial patterns of marks represent context-specific signals, termed a 'histone code'. It functions as one of the epigenetic regulatory mechanisms, which control reversible and heritable changes in cellular phenotype. Intermolecular models demonstrate thermodynamic benefits from multivalent engagement of nucleosomes, suggesting their widespread occurrence. However, so far only few multivalent readers are known and dissecting their function has been very challenging. This thesis focuses on HiMIDs with complex roles that simultaneously interact with two histone PTMs or two different substrates. Introducing the theoretical foundation, I discuss the thermodynamic and biological basis of how multivalent interactions can guide effector protein complexes, targeting their functions to distinct regions and chromatin states. Then, I present data from the characterisation of the readers DNMT3A-PWWP, DDX19A, and UHRF1-TTD in the context of multivalent engagement of histone PTMs and biomolecules. Starting with DNMT3A-PWWP, I quantified the binding of the wild-type (WT) and a mutant domain to histone H3K36me2/3 peptides, showing negligible differences, while my colleagues showed that the mutant has drastically reduced binding to DNA and nucleosomal substrates. I, then, studied the R-loop helicase DDX19A to demonstrate a very strong binding to H3K27me3 peptides in the nanomolar range, complementing the findings of a complex functional study. The latter showed that interaction with H3K27me3 is necessary for robust DDX19A-mediated R-loop resolution, and LSD1-target gene silencing. With UHRF1-TTD, I discovered and quantified its preferential binding to H3K4me1-K9me2/3 peptides vs H3K9me2/3 alone and engineered mutants with specific and differential binding changes leading to the discovery of a novel Kme1 read-out mechanism, based on the interaction of R207 methylene groups with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. High-throughput sequencing (HTS) data revealed strong TTD binding at chromatin sites with H3K4me1 peaks and broad H3K9me2/3 signal, which are enriched on enhancers and promoters of cell-type specific genes at the flanks of cell-type specific transcription factor binding sites. Data from the full-length protein in mouse and human cells evidenced the physiological role of the H3K4me1-K9me2/3 double marks in TTD-mediated UHRF1 recruitment. To further illustrate this point, I investigated UHRF1-dependent silencing of repeat elements (RE). To this end, I developed RepEnTools, improving the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, with carefully chosen and validated settings, enhancing accessibility, and adding some key functions. RepEnTools analyses showed that chromatin binding of hUHRF1-TTD and full-length mUHRF1 was strongly enriched on different REs promoters with the H3K4me1-K9me3 double mark where UHRF1 represses their expression. The data suggest a novel functional role for the H3K4me1-K9me3 signal of the histone code that is both sequence independent and conserved in two distinct mammals. Taken together, the work presented here is consistent with and supports the histone code theory, best illustrated by UHRF1-TTD which binds a specific double mark that has a biological meaning going beyond the meaning of the individual marks. In this thesis, I presented various mechanisms that influence epigenomic regulation, including chromatin 3D-architecture, accessibility, transcription factor recruitment, and chromatin marks. Especially in the context of UHRF1-TTD functions, I discussed how DNA, RNA, histones, and covalent modifications thereof interweave to produce the signalling network necessary throughout the lifetime of the mammalian cell, during differentiation, development and every other phase of life. Thus, within the three-dimensional scaffold of chromatin structures these biomolecules and their modifications collectively form the context-specific network of effectors and maintainers of the epigenomic modifications. The ways in which they influence transcription and translation are only now becoming unravelled. Hence, the recent data suggest the existence of not just a histone code, but a 3D-chromatin modification code, which dictates how biomolecules and their modifications collectively implement epigenomic regulation by interactions along the chromatin and through 3D space. As shown in these projects, readers commonly use the mechanism of multivalent interactions to interpret such contextual signals and guide epigenomic effectors to their targets. The tools and workflows that were developed and applied in this work can be employed to reveal more instances of refined read-out among HiMIDs. Additionally, I leveraged my experience with fluorescence spectroscopy and made contributions to another two published studies. The first study demonstrated that the DNMT3A-ADD Zn-finger domain, which is a known H3K4me0 reader, also binds to a domain from the MECP2 protein. The association was quantified, and the specificity demonstrated with a binding deficient triple mutant. This interaction offers complex additional regulation options to DNMT3A and MECP2, in interplay with the histone code. The second study focused on SETD2, a H3K36me3 depositing enzyme, and the mechanism of its preference for a designed “super substrate” peptide. By elegantly combining computational simulations and experimental data, the study demonstrated that an H3 peptide substrate predominantly exists in an extended conformation in solution, while the super substrate forms a hairpin conformation. Upon binding to the enzyme, the hairpin is opened and the super substrate adopts a similar conformation as the canonical substrate. These results highlighted the dynamic nature of solubilised peptides' conformations, their impact on protein-protein interactions, and the significance of dynamic conformational changes in interactions.
Open Access
Mechanistic basis of the increased methylation activity of the SETD2 protein lysine methyltransferase towards a designed super-substrate peptide
(2022) Schnee, Philipp; Choudalakis, Michel; Weirich, Sara; Khella, Mina S.; Carvalho, Henrique; Pleiss, Jürgen; Jeltsch, Albert
Protein lysine methyltransferases have important regulatory functions in cells, but mechanisms determining their activity and specificity are incompletely understood. Naturally, SETD2 introduces H3K36me3, but previously an artificial super-substrate (ssK36) was identified, which is methylated >100-fold faster. The ssK36-SETD2 complex structure cannot fully explain this effect. We applied molecular dynamics (MD) simulations and biochemical experiments to unravel the mechanistic basis of the increased methylation of ssK36, considering peptide conformations in solution, association of peptide and enzyme, and formation of transition-state (TS) like conformations of the enzyme-peptide complex. We observed in MD and FRET experiments that ssK36 adopts a hairpin conformation in solution with V35 and K36 placed in the loop. The hairpin conformation has easier access into the active site of SETD2 and it unfolds during the association process. Peptide methylation experiments revealed that introducing a stable hairpin conformation in the H3K36 peptide increased its methylation by SETD2. In MD simulations of enzyme-peptide complexes, the ssK36 peptide approached TS-like structures more frequently than H3K36 and distinct, substrate-specific TS-like structures were observed. Hairpin association, hairpin unfolding during association, and substrate-specific catalytically competent conformations may also be relevant for other PKMTs and hairpins could represent a promising starting point for SETD2 inhibitor development.
Open Access
The MECP2‐TRD domain interacts with the DNMT3A‐ADD domain at the H3‐tail binding site
(2022) Kunert, Stefan; Linhard, Verena; Weirich, Sara; Choudalakis, Michel; Osswald, Florian; Krämer, Lisa; Köhler, Anja R.; Bröhm, Alexander; Wollenhaupt, Jan; Schwalbe, Harald; Jeltsch, Albert
The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A‐ADD and MECP2‐TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull‐down, equilibrium peptide binding assays, and structural analyses. The region D529‐D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N‐terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2‐TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.
Open Access
Refined read‐out : the hUHRF1 Tandem‐Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3
(2023) Choudalakis, Michel; Kungulovski, Goran; Mauser, Rebekka; Bashtrykov, Pavel; Jeltsch, Albert
UHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem-Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1-K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read-out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. Individual TTD mutants showed up to a 10,000-fold preference for the double-modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1-K9me2 regions in human chromatin demonstrated in our TTD chromatin pull-down and ChIP-western blot data suggests that it has specific biological roles. Chromatin pull-down of TTD from HepG2 cells and full-length murine UHRF1 ChIP-seq data correlate with H3K4me1 profiles indicating that the H3K4me1-K9me2/3 interaction of TTD influences chromatin binding of full-length UHRF1. We demonstrate the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of cell-type-specific genes at the flanks of cell-type-specific transcription factor binding sites, and provided evidence supporting an H3K4me1-K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1-TTD binding to H3K4me1-K9me2/3 double marks in a cellular context.
Open Access
RepEnTools : an automated repeat enrichment analysis package for ChIP-seq data reveals hUHRF1 Tandem-Tudor domain enrichment in young repeats
(2024) Choudalakis, Michel; Bashtrykov, Pavel; Jeltsch, Albert
Background. Repeat elements (REs) play important roles for cell function in health and disease. However, RE enrichment analysis in short-read high-throughput sequencing (HTS) data, such as ChIP-seq, is a challenging task. Results. Here, we present RepEnTools , a software package for genome-wide RE enrichment analysis of ChIP-seq and similar chromatin pulldown experiments. Our analysis package bundles together various software with carefully chosen and validated settings to provide a complete solution for RE analysis, starting from raw input files to tabular and graphical outputs. RepEnTools implementations are easily accessible even with minimal IT skills (Galaxy/UNIX). To demonstrate the performance of RepEnTools , we analysed chromatin pulldown data by the human UHRF1 TTD protein domain and discovered enrichment of TTD binding on young primate and hominid specific polymorphic repeats (SVA, L1PA1/L1HS) overlapping known enhancers and decorated with H3K4me1-K9me2/3 modifications. We corroborated these new bioinformatic findings with experimental data by qPCR assays using newly developed primate and hominid specific qPCR assays which complement similar research tools. Finally, we analysed mouse UHRF1 ChIP-seq data with RepEnTools and showed that the endogenous mUHRF1 protein colocalizes with H3K4me1-H3K9me3 on promoters of REs which were silenced by UHRF1. These new data suggest a functional role for UHRF1 in silencing of REs that is mediated by TTD binding to the H3K4me1-K9me3 double mark and conserved in two mammalian species. Conclusions. RepEnTools improves the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, enhancing accessibility and adding some key functions. RepEnTools can analyse RE enrichment rapidly, efficiently, and accurately, providing the community with an up-to-date, reliable and accessible tool for this important type of analysis.