Browsing by Author "Tamas, Raluca"

Now showing 1 - 4 of 4

Open Access
The GEF‐H1/PKD3 signaling pathway promotes the maintenance of triple‐negative breast cancer stem cells
(2019) Lieb, Wolfgang S.; Lungu, Cristiana; Tamas, Raluca; Berreth, Hannah; Rathert, Philipp; Storz, Peter; Olayioye, Monilola A.; Hausser, Angelika
Open Access
H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1
(2017) Jurkowska, Renata Z.; Qin, Su; Kungulovski, Goran; Tempel, Wolfgang; Liu, Yanli; Bashtrykov, Pavel; Stiefelmaier, Judith; Jurkowski, Tomasz P.; Kudithipudi, Srikanth; Weirich, Sara; Tamas, Raluca; Wu, Hong; Dombrovski, Ludmila; Loppnau, Peter; Reinhardt, Richard; Min, Jinrong; Jeltsch, Albert
SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.
Open Access
Identification of protein lysine methylation readers with a yeast three-hybrid approach
(2018) Rawłuszko-Wieczorek, Agnieszka Anna; Knodel, Franziska; Tamas, Raluca; Dhayalan, Arunkumar; Jeltsch, Albert
Background: Protein posttranslational modifications (PTMs) occur broadly in the human proteome and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylations sites among histone and non-histone proteins have been characterized, however, the list of readers associated with them is incomplete. Results: This study introduces a modified yeast-three-hybrid system (Y3H) to screen for methyl-lysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like chromodomain of CBX1 or MPP8 as prey. We demonstrate the proof of principle of the method, and, more importantly, we show that an unbiased screen using a library composed of human-specific open reading frames led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei. Conclusions: Our approach introduces a cost-effective method for screening reading domains binding to histone and non-histone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the high capability of the Y3H assay which will allow for proteome wide screens of PTM readers.
Open Access
Investigation of proteins responsible for the establishment and recognition of prominent lysine modifications
(2014) Tamas, Raluca; Jeltsch, Albert (Prof. Dr.)
Histone post-translational modifications influence chromatin architecture, either by direct effects on the interaction between histones and DNA, or indirectly, by serving as docking places for regulatory proteins, which bind through conserved functional domains termed “reading” domains. Different combinations of histone modifications define various chromatin states, each of which being associated with a particular set of regulatory enzymes. Lysine methylation is an important histone post-translational modification, which can occur at various positions in histones, with different roles in epigenetic regulation. This mark is generally established by SET domain Protein Lysine Methyltransferases (PKMTs). Recently, PKMTs have been reported to also methylate numerous non-histone substrates, which subsequently recruit so called “reading” domains. These domains specifically interact with the methylated lysine in a sequence context-dependent manner. In this work, I tried to establish a Yeast-3-Hybrid method for the identification of methylation-dependent interactors of methylated non-histone proteins. For validation, I attempted to test the interaction between reported PKMT substrates fused to the Gal4-DNA-Binding Domain and methyl-“readers” fused to the Gal4-Activation Domain in yeast, either in the presence or absence of the corresponding PKMTs. Later in the project the known “reading” domains would be replaced by a library of human cDNA, in order to search for novel “readers” of protein lysine methylation marks. Additionally, this work presents the study of the substrate specificities of two SET domain methyltransferases responsible for the methylation of histone 3 lysine 4 (H3K4), which are mutually exclusive members of the same coactivator complex, the human COMPASS. In this study, SET1A, an H3K4 trimethylase, was shown to be active only as part of the core COMPASS complex. This PKMT proved to have a higher preference for some sequences other than histone 3, justifying a search for novel non-histone substrates. MLL2, a member of the mixed lineage leukemia (MLL) family, responsible for H3K4 monomethylation, revealed stimulation of activity when part of the core COMPASS complex, and showed some differences in the substrate specificity when acting alone, compared to the complex. The search for non-histone protein substrates is in progress for SET1A/COMPASS, and also MLL2 alone and within the complex. The targeting of most PKMTs is achieved with the help of histone modification “reading” or DNA-binding domains. The binding specificity of the PHD finger “reading” domains of MLL2, and its paralog MLL3, was investigated during this doctoral study. Although most of the PHD fingers did not bind to histone tails, the MLL2 PHD 3-5 group of domains and the MLL3 PHD 4-6 group of domains bound specifically to modified histone tail peptides. Preference towards both histone 3 (H3) and histone 4 (H4) was identified and the strongest binding was seen on H4 peptides containing acetylation at lysine 16 together with multiple acetylations or methylations. This finding suggested recruitment to active chromatin, which is enriched in acetylation marks, but the specificity needs to be further confirmed and characterized in more detail. I also investigated the histone binding specificity of PHF1, a member of the Polycomb Repressive Complex 2. This complex is responsible for developmental gene repression by the trimethylation of histone 3 lysine 27 (H3K27me3). The tudor domain of PHF1 showed preferred binding to its target, H3K27me3 in the sequence context of testis-identified histone variant H3T, in comparison to the canonical histone H3.1. The specificity for the same mark and histone variant was also identified for the chromodomain of the Polycomb Repressive Complex 1 member, CBX7, while the chromodomain of its paralog, CBX2, did not show discrimination between the histone variants, although it presented the same specificity towards the H3K27me3 mark. We propose that the discrimination between histone variants is a unique feature of some “reading” domains, and the role of this particular function needs to be elucidated. Moreover, the H3K27me3-specific CBX7 chromodomain was used as a tool in the validation of new methods developed by Kungulovski et al., 2014, with the purpose of replacing antibodies raised against specific histone modifications in adaptations of several antibody-based assays. Finally, this PhD work also presents the binding specificity of the chromodomain of the SUV39H1 methyltransferase. SUV39H1 is responsible for histone 3 lysine 9 trimethylation (H3K9me3), and the consequent gene repression and silencing of heterochromatin. I showed that the chromodomain of SUV39H1 bound specifically to H3K9me3, and binding of the chromodomain to its target peptide seemed to inhibit the catalytic activity of the enzyme in our in vitro conditions.