Evan J. Worden

Bacterial pathogens modulate host cell physiology by secreting effector proteins that rewire host signaling pathways. A subset of these effectors directly modify host chromatin to reprogram gene expression and promote infection. While these enzymes are thought to function autonomously, the extent to which the host epigenetic landscape regulates their activity remains largely unknown. RomA and its homolog LegAs4 are Set domain-containing lysine methyltransferases from Legionella pneumophila that methylate histone H3 at lysine 14 (H3K14) to suppress host immune responses and enhance intracellular bacterial replication. Here, we demonstrate that RomA activity is constrained by preexisting host histone posttranslational modifications (PTMs) through multiple layers of histone PTM cross talk. RomA selectively binds and methylates unmodified histone H3 tails and is inhibited by histone PTMs associated with active transcription, including H3K4 trimethylation, H3K4 acetylation, and H4K12 mono-methylation. We identify both cis- and trans-histone regulatory mechanisms, whereby unmodified H3K4 and H3K14 must reside on the same H3 tail to support RomA activity, while H4K12me1 inhibits RomA across the nucleosome. Notably, cryo-electron microscopy analysis and biochemical data reveal that RomA does not engage the nucleosome acidic patch but instead associates flexibly through histone tails. Together, these findings establish the host epigenetic regulation of bacterial effectors as a fundamental and previously unrecognized layer of host–pathogen interactions.

Bacterial pathogens modulate host cell physiology by secreting effector proteins that rewire host signaling pathways. A subset of these effectors directly modify host chromatin to reprogram gene expression and promote infection. While these enzymes are thought to function autonomously, the extent to which the host epigenetic landscape regulates their activity remains largely unknown. RomA and its homolog LegAs4 are Set domain-containing lysine methyltransferases from Legionella pneumophila that methylate histone H3 at lysine 14 (H3K14) to suppress host immune responses and enhance intracellular bacterial replication. Here, we demonstrate that RomA activity is constrained by pre-existing host histone post-translational modifications (PTMs) through multiple layers of histone PTM crosstalk. RomA selectively binds and methylates unmodified histone H3 tails and is inhibited by histone PTMs associated with active transcription, including H3K4 trimethylation, H3K4 acetylation, and H4K12 mono-methylation. We identify both cis- and trans-histone regulatory mechanisms, whereby unmodified H3K4 and H3K14 must reside on the same H3 tail to support RomA activity, while H4K12me1 inhibits RomA across the nucleosome. Notably, cryo-EM analysis and biochemical data reveal that RomA does not engage the nucleosome acidic patch but instead associates flexibly through histone tails. Together, these findings establish the host epigenetic regulation of bacterial effectors as a fundamental and previously unrecognized layer of host-pathogen interactions. SIGNIFICANCE: Bacterial pathogens reprogram host gene expression by delivering effector proteins that modify chromatin, but it is not known how the host epigenetic environment impacts effector function. Here, we show that the Legionella effector RomA senses and responds to the host’s existing epigenetic landscape and is selectively active only in specific chromatin contexts through mechanisms resembling those used by eukaryotic chromatin regulators. Notably, we uncover that RomA utilizes cis-histone and trans-histone crosstalk mechanisms previously observed only in eukaryotic systems. These reveals an unexpected form of host-pathogen crosstalk in which bacterial effector activity can be constrained by host epigenetic modifications.

H/ACA small nucleolar ribonucleoproteins (H/ACA snoRNPs) facilitate essential cellular processes such as RNA modification, folding, and stability. Here, we present multiple cryo-EM structures of endogenous insect H/ACA snoRNPs containing two protomers assembled on a two-hairpin H/ACA snoRNA. By characterizing key protein–protein and protein–RNA interactions, we reveal the coordination of pseudouridylation activity across the two protomers which explains the predominance of two-hairpin structures in eukaryotic H/ACA snoRNAs. Moreover, we found that several mutations in H/ACA proteins associated with dyskeratosis congenita (DC) directly impair pseudouridine formation suggesting how these mutations disrupt RNA modification and ribosome biogenesis in this disease. Additionally, we uncover coordinated structural changes between Nop10, Nhp2, and the N-terminal extensions of Cbf5 in the 3′ protomer that resemble active and inactive conformations and may regulate H/ACA snoRNP activity. In summary, this study provides detailed insight into the structure and function of RNA modification-competent H/ACA snoRNPs, which play pivotal roles in cellular processes including ribosome biogenesis, rRNA folding, (m)RNA modification, and telomere maintenance.

De novo DNA methylation is mediated by DNA methyltransferases DNMT3A and DNMT3B, in cooperation with the catalytically inactive paralogs DNMT3L and DNMT3B3. DNMT3L is predominantly expressed in embryonic stem cells to establish methylation patterns and is silenced upon differentiation, with DNMT3B3 substituting in somatic cells. Here we present high-resolution cryo-electron microscopy structures of nucleosome-bound, full-length DNMT3A2–3L and its oligomeric assemblies in the nucleosome-free state. We identified the critical role of DNMT3L as a histone modification sensor, guiding chromatin engagement through a mechanism distinct from DNMT3B3. The structures show a 180° rotated ‘switching helix’ in DNMT3L that prevents direct interaction with the nucleosome acidic patch. Instead, nucleosome binding is mediated by the DNMT3L ADD domain, while the DNMT3A PWWP domain exhibits reduced engagement in the absence of H3K36 methylation. The oligomeric arrangement of DNMT3A2–3L in nucleosome-free states highlights its dynamic assembly and potential allosteric regulation. We further capture dynamic structural movements of DNMT3A2–3L on nucleosomes. These findings uncover a previously unknown mechanism by which DNMT3A–3L mediates de novo DNA methylation on chromatin through complex assembly, histone tail sensing, dynamic DNA search and regulated nucleosome engagement, providing insights into epigenetic regulation.

De novo DNA methylation is a fundamental epigenetic process essential for early development. It is mediated by DNA methyltransferase DNMT3A and DNMT3B, in cooperation with catalytically inactive DNMT3L. Abnormal DNA methylation contributes to genomic instability and oncogenesis, yet the mechanisms governing its establishment and regulation remain unclear. Here, we present high-resolution cryo-EM structures of the nucleosome-bound full-length DNMT3A2-3L and its oligomeric assemblies in the nucleosome-free state. Strikingly, DNMT3L C-terminal “Switching Helix” displayed a distinct conformation with a 180° rotational difference compared to its DNMT3B3 counterpart in the presence of nucleosome, preventing direct interactions with the nucleosome acidic patch. Instead, DNMT3L ADD domain promotes nucleosome binding, while DNMT3A PWWP domain inhibits it, indicating multi-layer regulation. Additionally, the novel oligomeric arrangement of DNMT3A2-3L in nucleosome-free states highlights its dynamic assembly and potential allosteric regulation. Furthermore, we identified the critical role of DNMT3L as a sensor of histone modifications, guiding the complex’s chromatin interaction. These findings uncover a previously unknown mechanism by which DNMT3A-3L mediates de novo DNA methylation on chromatin through complex reorganization, histone tail sensing, dynamic DNA search, and nucleosome engagement, providing key insights into epigenetic regulation.

: Histone H3K9 methylation (H3K9me) by Setdb1 silences retrotransposons (rTE) by sequestering them in constitutive heterochromatin. Atf7IP is a constitutive binding partner of Setdb1 and is responsible for Setdb1 nuclear localization, activation and chromatin recruitment. However, structural details of the Setdb1/Atf7IP interaction have not been evaluated. We used Alphafold2 predictions and biochemical reconstitutions to show that one copy of Setdb1 and two copies of Atf7IP form a hetero-trimeric complex in vitro and in cells . We also find that Atf7IP self-associates, forming multimeric complexes that are resolved upon Setdb1 binding. Setdb1 binds to Atf7IP through coiled coil interactions that include both Setdb1 nuclear export signals (NES). Atf7IP directly competes with CRM1 to bind the Setdb1 NES motifs, explaining how Atf7IP prevents CRM1-mediated nuclear export of Setdb1. Setdb1 also forms hetero-trimeric complexes with the Atf7IP paralog Atf7IP2 and we show that Setdb1 can form mixed heterotrimers comprising one copy of each Setdb1, Atf7IP and Atf7IP2. Atf7IP and Atf7IP2 are co-expressed in many tissues suggesting that heterotrimers with different compositions of Atf7IP and Atf7IP2 may differentially regulate H3K9me by fine-tuning Setdb1 localization and activity.

De novo cytosine methylation is essential for mammalian development and is deposited by DNMT3A and DNMT3B. In cells, DNA methylation occurs in the context of chromatin, where nucleosomes are connected by DNA linkers. Here, we report Cryo-EM structures of DNMT3A2/3B3 bound to di-nucleosomes with different linker lengths. We show that DNMT3A2/3B3 preferentially binds di-nucleosomes separated by short DNA linkers by inducing large-scale changes to the di-nucleosome structure, enabling each DNMT3B3 subunit to bind each nucleosome. Linker length and the position of cytosines within the linker control DNA methylation, indicating that a significant fraction of linkers in chromatin are naturally resistant to DNMT3A2/3B3 activity. Finally, DNMT3A2/3B3 scans for H3K36me2-3 modifications, explaining how H3K36 methylation simulates DNMT3A2 activity. Our structure is the first example of a DNA methyltransferase interacting with higher-order nucleosome substrates and provides new insights on how DNA methylation takes place in chromatin.