Browsing by Author "Kungulovski, Goran"

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Open Access
Application of dual reading domains as novel reagents in chromatin biology reveals a new H3K9me3 and H3K36me2/3 bivalent chromatin state
(2017) Mauser, Rebekka; Kungulovski, Goran; Keup, Corinna; Reinhardt, Richard; Jeltsch, Albert
Histone post-translational modifications (PTMs) play central roles in chromatin-templated processes. Combinations of two or more histone PTMs form unique interfaces for readout and recruitment of chromatin-interacting complexes, but the genome-wide mapping of co-existing histone PTMs remains an experimentally difficult task. We introduce here a novel type of affinity reagents consisting of two fused recombinant histone modification interacting domains (HiMID) for direct detection of doubly modified chromatin. To develop the method, we fused the MPP8 Chromodomain and DNMT3A PWWP domain which have a binding specificity for H3K9me3 and H3K36me2/3, respectively. We validate the novel reagent biochemically and in ChIP applications and show its specific interaction with H3K9me3-H3K36me2/3 doubly modified chromatin. Modification specificity was confirmed using mutant double-HiMIDs with inactivated methyllysine binding pockets. Using this novel tool, we mapped co-existing H3K9me3-H3K36me2/3 marks in human cells by chromatin interaction domain precipitation (CIDOP). CIDOP-seq data were validated by qPCR, sequential CIDOP/ChIP and by comparison with CIDOP- and ChIP-seq data obtained with single modification readers and antibodies. The genome-wide distribution of H3K9me3-H3K36me2/3 indicates that it represents a novel bivalent chromatin state, which is enriched in weakly transcribed chromatin segments and at ZNF274 and SetDB1 binding sites.
Open Access
Development and application of experimental tools for studying the distribution and dynamics of chromatin modifications
(2015) Kungulovski, Goran; Jeltsch, Albert (Prof. Dr.)
All cells in a multicellular organism carry the same genetic information, and yet throughout their lifetimes they follow unique transcriptional programs, which lead to phenotypical and functional differences. These differential gene expression programs are enacted by highly coordinated epigenetic mechanisms, which include modifications of chromatin, such as DNA methylation and histone post-translational modifications. Their involvement in chromatin-associated processes, association with different genomic elements and the means of their establishment and maintenance are crucial scientific issues. The primary focus of this study was to shed light on the genome-wide distribution of chromatin modifications and the effects of their local establishment. First, we focused our efforts into developing and applying novel affinity reagents for local and genome-wide characterization of histone modifications. We made use of native and engineered recombinant proteins that have an intrinsic ability to interact specifically with modified histones. In a rigorous side-by-side comparison with high quality histone modification antibodies, we successfully applied these novel affinity reagents in approaches such as western blot and chromatin precipitation coupled with quantitative PCR or massively parallel sequencing, following established quality control criteria. By this, we validated the feasibility of this strategy. We also discussed in detail the advantages of using recombinant proteins in lieu of antibodies, such as their cheap production with high yield, ease of protein engineering and consistent quality and reproducibility. Secondly, we wanted to clarify some of the principal mechanisms by which epigenetic modifications operate inside the cell. To this aim, we established and applied an approach based on zinc finger targeted promoter methylation of VEGF-A (vascular endothelial growth factor) in order to study the in vivo effects and dynamics of histone modifications and DNA methylation. Adenoviral constructs made of the targeting zinc finger were fused with catalytic domains from epigenetic enzymes such as DNA and histone methyltransferases and were used to infect cells. By these means we were able to successfully follow in detail the establishment, effects and kinetics of the installed chromatin modifications. Our data indicate that local chromatin editing of a target locus can change its intial chromatin state and consequently modulate its transcriptional output, albeit for a short period of time, before it returns to its native configuration. In conclusion, in this body of work we were able to successfully develop and apply novel affinity reagents for studying the distribution of histone modifications. Along the same lines, we also successfully developed and applied a strategy for targeted chromatin editing, which allowed us to study the dynamics of establishment, downstream effects and maintenance of chromatin modifications.
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
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
Targeted epigenome editing of an endogenouslocus with chromatin modifiers is not stably maintained
(2015) Kungulovski, Goran; Nunna, Suneetha; Thomas, Maria; Zanger, Ulrich M.; Reinhardt, Richard; Jeltsch, Albert
Background: DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter. Results: Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression Levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment. Conclusions: The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary.