Epigenetic regulation of genome function relies on the dynamic interplay between DNA and histone modifications and chromatin-binding proteins. Most genomic loci harbor multiple modifications that do not act in isolation but instead form interconnected regulatory patterns. Despite significant progress in elucidating the roles of individual modifications, much less is known about how modifications within such composite patterns cooperate to orchestrate specific genomic processes. My doctoral and postdoctoral research contributed to addressing this fundamental question by exploring the combinatorial complexity of histone modification landscapes and investigating the mechanisms through which nuclear protein machineries decode composite modification patters.

Quantitative proteomic analysis of combinatorial histone modifications

Histone proteins undergo a diverse array of post-translational modifications at specific residues, creating complex combinatorial patterns that regulate chromatin structure and function. However, the precise combinations present in chromatin in vivo remain poorly characterized, largely due to limitations in existing methodologies. Commonly used techniques, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq), are restricted in both the range of modifications they can probe and their ability to resolve co-occurring marks. To overcome these challenges, I focused my doctoral research in Ole Jensen’s lab on developing and applying mass spectrometry (MS)-based proteomics approaches for the comprehensive quantitative profiling of combinatorial histone modifications. My work provided pioneering insights into the molecular architecture of histone modification landscapes across different cell types and organisms, including Trypanosoma brucei, humans, and mice. Using a middle-down MS approach, I identified and quantified hundreds of combinatorial histone modifications present in mammalian cells in vivo. Remarkably, the N-terminal tail of histone H3 alone was, on average, decorated by 2 to 4 modifications and could simultaneously bear up to 7 distinct marks. Intriguingly, modifications traditionally associated with either active or repressive chromatin were often found co-existing on the same H3 tail, challenging established paradigms and emphasizing the unresolved complexity of epigenetic regulation. These modifications did not co-occur randomly but instead formed highly defined patterns, pointing to functional cross-talk where one modification could influence the addition or removal of others. My work advanced our understanding of complex histone modification patterns, laying a critical foundation for uncovering their regulatory roles in genome organization and function.

–  Maree J.P, Tvardovskiy A, Ravnsborg T, Jensen O.N, Rudenko G, Patterton H.G. Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A, Nucleic Acids Research, 2022.

–  Tvardovskiy A, Wrzesinski K, Sidoli S, Fey S.J, Rogowska-Wrzesinska A, Jensen O.N., Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns, Mol Cell Proteomics, 2015.

–  Tvardovskiy A, Schwammle V, Kempf S.J, Rogowska-Wrzesinska A, Jensen O.N., Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape, Nucleic Acids Research, 2017.

Lifelong reorganization of histone modification landscape

Epigenetic changes are increasingly recognized as integral to the aging process, with growing evidence linking them to the progressive decline in cellular and organismal function. While much attention has been given to the reorganization of DNA methylation patterns over time, the age-dependent dynamics of the histone modification landscape remain poorly characterized. To address this, I applied middle-down MS-based proteomics to quantitatively profile individual and combinatorial H3 modifications across multiple tissues throughout the lifespan of mice. The results revealed that the genome-wide abundances of multiple histone modifications change progressively and non-randomly with age. Some alterations were tissue-specific, while others—such as a consistent increase in H3 lysine 36 methylation and changes in several combinatorial H3 modification patterns—were observed broadly across tissues, suggesting shared processes underlying chromatin remodeling with age. Interestingly, these changes were accompanied by the gradual accumulation of the non-canonical histone variant H3.3, which by late adulthood had almost entirely replaced the canonical H3.1 and H3.2 isoforms. The distinct modification profile of H3.3, compared to its canonical counterparts, suggests that age-dependent histone variant exchange may play a causal role in reshaping histone modification patterns over time. My work provided the first comprehensive quantitative analysis of dynamic changes in histone H3 modifications and variants, offering insights into potential mechanisms underlying chromatin reorganization during aging.

–  Tvardovskiy A, Schwammle V, Kempf S.J, Rogowska-Wrzesinska A, Jensen O.N., Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape, Nucleic Acids Research, 2017.

Decoding the language of epigenetic modifications

DNA and histone modifications regulate genome function primarily by modulating the binding of nuclear proteins to chromatin. Nucleosomes carry diverse arrays of modifications, while many chromatin-associated proteins contain multiple modification-binding domains, suggesting that recognizing composite modification signatures is central to genome regulation. However, despite advances in identifying ‘readers’ of individual modifications, how nuclear proteins interpret complex modification landscapes remains largely unclear.

During my postdoctoral work in Till Bartke’s lab, I collaborated with Dr. Lukauskas and Dr. Nguyen to explore how combinatorial modification patterns influence protein binding to chromatin in vitro. To address this, we created a library of semi-synthetic dinucleosomes incorporating various modification signatures representative of promoter, enhancer, and heterochromatin states. Using these dinucleosomes as baits in affinity purification experiments, we systematically analyzed interactions between the human nuclear proteome and distinct chromatin states via mass spectrometry. We quantitatively described the chromatin-binding behaviors of several hundred proteins and identified sets of modifications that either positively or negatively regulate their association with nucleosomes. Our findings revealed that the binding of numerous factors, including transcription activators, repressors, and chromatin remodelers, is synergistically regulated by multiple interconnected modifications. Notably, modifications characteristic of distinct chromatin states varied greatly in their regulatory potential. For example, promoter-associated trimethylation of lysine 4 on histone H3 and hyperacetylated H3 and H4 tails profoundly influenced the binding of many proteins, while enhancer-associated acetylation of lysine 27 and monomethylation of lysine 4 appeared largely inert in targeting proteins to chromatin. Our work provided the first systematic characterization of how combinatorial chromatin modification patterns are decoded by the nuclear proteome, offering numerous insights into the complex regulatory circuits orchestrating key genomic processes.

– Tvardovskiy A, Nguyen NV, Bartke T. Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spectrometry, Methods Mol Biol., 2022

–  Lukauskas S*, Tvardovskiy A*, Nguyen N.V*, Stadler M, Faull P, Ravnsborg T, Aygenli B.Ö, Dornauer S, Flynn H, Lindeboom R.G.H, Barth T.K, Brockers K, Hauck S.M, Vermeulen M, Snijders A.P, Müller C.L, DiMaggio P.A, Jensen O.N, Schneider R, Bartke T. Decoding chromatin states by proteomic profiling of nucleosome readers, Nature, 2024.  * These authors contributed equally to this work.

– Tvardovskiy A, Lukauskas S. Decoding the language of chromatin modifications with MARCS, Nat Rev Genet., 2024