Biology subjects

Worden, E. J.

Publications and source records attributed to Worden, E. J..

10 recordsLinked to original sources

bioRxiv2026

A feed-forward UHRF1 read-write mechanism supports H3 multi- mono-ubiquitination and DNA methylation maintenance at CpG-sparse regions

The epigenetic inheritance of mammalian DNA methylation requires DNMT1 and its E3 ligase cofactor UHRF1. At newly replicated chromatin, UHRF1 recognition of hemi-methylated DNA and histone H3 N-terminal tails directs catalysis of H3K14, H3K18, and/or H3K23 mono-ubiquitination to recruit DNMT1. While it is appreciated that UHRF1 can deposit multiple mono-ubiquitin marks on a single H3 tail and that DNMT1 recognizes this state through tandem ubiquitin interacting motifs, the mechanism that promotes successive ubiquitination and the biological function of multi-mono-ubiquitination are unknown. Here, we show that UHRF1 directly binds its mono-ubiquitinated H3 products through a previously uncharacterized LGDDSL loop in Tudor 2 of its tandem Tudor domain (TTD) to promote further ubiquitin deposition. Disruption of this ubiquitin reading activity impairs H3 multi-mono-ubiquitination and accelerates DNA methylation loss within late-replicating, CpG-sparse genomic regions that are characteristic of partially methylated domains (PMDs) in cancer and aging cells. These methylation defects overlap those observed by disruption of UHRF1 ubiquitin ligase activity, providing convergent evidence that both writing and reading of H3 ubiquitination support CpG-sparse DNA methylation maintenance. Together, these findings establish a feed-forward ubiquitin read-write mechanism that generates multi-mono-ubiquitinated H3 and safeguards DNMT1-dependent DNA methylation maintenance at vulnerable genomic regions of the mammalian methylome.

molecular biology↗

bioRxiv2026

Histone modification crosstalk between host and pathogen

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. SIGNIFICANCEBacterial 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 hosts 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.

biochemistry↗

bioRxiv2026

Parkinson's disease-associated LRRK2 risk variant, G2385R, enhances Rab substrate phosphorylation and impairs neuronal integrity

Mutations in the LRRK2 gene are the most frequent cause of familial Parkinsons disease (PD) whereas common variants are associated with an increased risk for sporadic PD. LRRK2 encodes a multi-domain protein displaying two functional enzymatic activities: GTPase and kinase. Familial LRRK2 mutations have been linked to alterations in its GTPase and kinase activities, elevated substrate phosphorylation, as well as increased neurotoxicity in cell and animal models. In addition to familial mutations, several common LRRK2 coding variants have been associated with PD risk in different ethnic populations. Little is known about how these coding variants modulate the risk of developing PD. In this study, we aim to evaluate whether a collection of LRRK2 coding risk variants (A419V, N551K, R1398H, R1628P, M1646T, S1647T, G2385R) modify the biochemical properties of the LRRK2 protein and LRRK2-mediated neurotoxicity using cell-based assays. With the exception of G2385R, we find that coding risk variants have minimal impact on LRRK2 steady-state protein levels, GTP-binding, phosphorylation at Ser910, Ser935, and Ser1292, and subcellular localization in human cell lines and cultured primary neurons. G2385R reduces the steady-state levels of LRRK2 and exhibits reduced phosphorylation at Ser910/Ser935 and Ser1292, consistent with diminished kinase activity. Notably, however, LRRK2 variants associated with increased PD risk (A419V, R1628P, M1646T, and G2385R) significantly elevate the levels of LRRK2-mediated Rab10 phosphorylation by two-fold in cells. In contrast, LRRK2 variants associated with reduced PD risk (N551K, R1398H, and N551K-R1398H) do not alter normal LRRK2-mediated pRab10 levels. Intriguingly, we find that both PD-risk and PD-protective LRRK2 variants can respond normally to kinase activation by different lysosomal stressors (chloroquine, nigericin and monensin) to robustly induce pRab10 levels in cells. The PD risk variant, G2385R LRRK2, significantly inhibits neurite outgrowth in primary cortical neurons compared to wild-type LRRK2. Combining each coding risk variant with the familial G2019S mutation has minimal impact on the elevated kinase activity of G2019S LRRK2 or its capacity to inhibit neurite outgrowth. Our study indicates that LRRK2-dependent Rab phosphorylation represents a relevant readout of PD risk induced by LRRK2 coding variants and demonstrates that the PD risk variant, G2385R LRRK2, creates a hyperactive kinase that can impair neuronal integrity at comparable levels to the effects of G2019S LRRK2.

neuroscience↗

bioRxiv2025

Molecular Mechanisms of DNMT3A-3L-Mediated de novo DNA Methylation on Chromatin

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{degrees} 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 complexs 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.

molecular biology↗

bioRxiv2025

Interprotomer Communication and Functional Asymmetry in H/ACA snoRNPs

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, catalytically active 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 discovered that several mutations in H/ACA proteins associated with Dyskeratosis congenita 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, asymmetric H/ACA snoRNPs, which play pivotal roles in cellular processes including ribosome biogenesis, rRNA folding, (m)RNA modification, and telomere maintenance. Significance statementH/ACA snoRNPs are critical for numerous RNA processes as they utilize different H/ACA snoRNAs to introduce pseudouridines in ribosomal and other non-coding RNAs as well as mRNAs. In addition, H/ACA snoRNPs stabilize and fold RNA thereby contributing to ribosome biogenesis and telomere maintenance. Mutations in H/ACA snoRNP subunits such as dyskerin (pseudouridine synthase), Nhp2, and Nop10 cause the genetic disorder Dyskeratosis congenita. Here, we present cryo-EM structures of active eukaryotic H/ACA snoRNPs, revealing an asymmetric, dimeric complex of two protomers. We identify the key inter-protomer contact hotspots and biochemically characterize mutations at these interfaces. Our findings emphasize the importance of inter-subunit interactions for snoRNP stability and activity, providing mechanistic insight into H/ACA snoRNP function and Dyskeratosis congenita pathogenesis.

biochemistry↗

bioRxiv2024

Setdb1 and Atf7IP form a hetero-trimeric complex that blocks Setdb1 nuclear export

AbstractHistone 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.

biochemistry↗

bioRxiv2024

The structural basis for de novo DNA methylation in chromatin

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.

biophysics↗

bioRxiv2022

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

The human Mixed Lineage Leukemia-1 (MLL1) complex orchestrates methylation of histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variation in the docking of MLL1-WRAD on nucleosomes and left ambiguous portions of Ash2L and the position of DPY30. We used an integrated approach combining cryo-electron microscopy and mass spectrometry-crosslinking to determine structures of the MLL1-WRAD complex bound to ubiquitinated nucleosomes containing the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The subunits in all four states are positioned on the nucleosome in manner that is similar to a previous structure of MLL1-WRAD bound to ubiquitinated nucleosome, but that differs from structures with unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex. SignificanceThe Mixed Lineage Leukemia-1 (MLL1) complex plays a role in activating transcription by methylating lysine 4 in histone H3, a reaction that is stimulated by the presence of ubiquitin conjugated to histone H2B. Recent structures of the core MLL1 complex, termed MLL1-WRAD, have revealed the existence of multiple docking states and have also left ambiguous portions of the structure. Here we combine mass spectrometry-cross linking with cryo-EM to model additional regions of the MLL1-WRAD complex and identify a series of states that light on complex assembly and the role that ubiquitin plays in orienting MLL1-WRAD on nucleosomes.

biophysics↗

bioRxiv2019

Structural basis for COMPASS recognition of an H2B-ubiquitinated nucleosome

Methylation of histone H3K4 is a hallmark of actively transcribed genes that depends on mono-ubiquitination of histone H2B (H2B-Ub). H3K4 methylation in yeast is catalyzed by Set1, the methyltransferase subunit of COMPASS. We report here the cryo-EM structure of a six-protein core COMPASS subcomplex, which can methylate H3K4 and be stimulated by H2B-Ub, bound to a ubiquitinated nucleosome. Our structure shows that COMPASS spans the face of the nucleosome, recognizing ubiquitin on one face of the nucleosome and methylating H3 on the opposing face. As compared to the structure of the isolated core complex, Set1 undergoes multiple structural rearrangements to cement interactions with the nucleosome and with ubiquitin. The critical Set1 RxR motif adopts a helix that mediates bridging contacts between the nucleosome, ubiquitin and COMPASS. The structure provides a framework for understanding mechanisms of trans-histone cross-talk and the dynamic role of H2B ubiquitination in stimulating histone methylation.

molecular biology↗

bioRxiv2018

The mechanism of cross-talk between histone H2B ubiquitination and H3 methylation by Dot1L

Methylation of histone H3, lysine 79 (H3K79), by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B at lysine 120 (H2B-Ub), and is a well-characterized example of histone modification cross-talk that is conserved from yeast to humans. The mechanism by which H2B-Ub stimulates Dot1L to methylate the relatively inaccessible histone core H3K79 residue is unknown. The 3.0 [A] resolution cryo-EM structure of Dot1L bound to ubiquitinated nucleosome reveals that Dot1L contains binding sites for both ubiquitin and the histone H4 tail, which establish two regions of contact that stabilize a catalytically competent state and positions the Dot1L active site over H3K79. We unexpectedly find that contacts mediated by both Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to project deep into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals an unexpected structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.

biochemistry↗