Biology subjects

Kasinath, V.

Publications and source records attributed to Kasinath, V..

12 recordsLinked to original sources

bioRxiv2026

Structures of MeCP2 bound to nucleosomes reveal distinct mechanisms of Rett syndrome mutations

Methyl-CpG binding protein 2 (MeCP2) is a chromatin-associated regulator essential for neuronal gene regulation, and pathogenic mutations in MeCP2 cause Rett syndrome (RTT). However, the mechanisms governing MeCP2 engagement on chromatin and the effects of RTT mutations on nucleosome interactions remain poorly understood. We determined cryo-EM structures of MeCP2 bound to mono-nucleosomes with or without linker DNA methylation. In the absence of linker DNA methylation, the methyl-CpG binding domain (MBD) engages nucleosomal DNA near superhelical location +/- 7, whereas a methylated linker CpG redirects MBD to the linker methylation site. Quantitative EMSA, MNase footprinting, and fluorescence polarization show that both MBD and AT-hook regions cooperate to stabilize nucleosome binding, and that six common RTT variants (R133C, T158M, R306C, R168X, R255X, R270X) fall into four mechanistic classes. Loss-of-function truncations progressively weaken binding, whereas missense variants show near-WT DNA affinity. Among the variants, R133C shows near wild-type affinity for naked DNA but loses methylation-directed nucleosome engagement. Our analysis of human neuronal transcriptomic and chromatin occupancy datasets showed that RTT variants R133C and R168X lose CpG-island specificity and redistribute in the genome through distinct biochemical routes. Together, this work establishes a framework linking variant-specific defects in nucleosome binding to chromatin-targeting failure and transcriptional dysregulation in RTT.

biochemistry↗

bioRxiv2026

Structures of pUG-fold RNA bound to DNMT1 reveal a mechanism for RNA-mediated epigenetic regulation

Many chromatin-associated proteins have been found to bind RNA as a means of epigenetic regulation. Specifically, DNA methyltransferase 1 (DNMT1), which maintains cytosine methylation at CpG dinucleotides, is inhibited by RNA at transcribed DNA loci in cells. However, the mechanisms by which RNA binds DNMT1 and inhibits its activity remain unknown. Here, we determine a series of cryogenic electron microscopy (cryo-EM) structures of human DNMT1 bound to pUG-fold RNA, a non-canonical G-quadruplex previously observed to inhibit activity, revealing two distinct RNA-binding modes. The pUG-fold RNA binds the surface of DNMT1 in its autoinhibited conformation across a positively charged surface between the methyltransferase domain and the CXXC domain, and it binds directly in the active site of an open DNMT1 conformation. RNA binding is sterically incompatible with substrate DNA engagement in both states. Our 2.5 [A] structure captures the intricate network of hydrogen bonds and electrostatic interactions between amino acids in the methyltransferase domain and the tetrad layers of pUG-fold RNA. Metadynamics molecular dynamics simulations provide an orthogonal view of the conformational landscape of DNMT1, revealing the two distinct RNA-binding modes. Furthermore, our analysis of published DNMT1 RIP-seq and eCLIP-seq data confirms that DNMT1-interacting RNAs in cells exhibit a strong propensity to form non-canonical G-quadruplex RNA structures. Collectively, our study provides the first structural basis for pUG-fold RNA recognition by a protein and illustrates how cryo-EM and AI-based methods for protein and RNA structure prediction synergize to inform the mechanism of RNA-mediated regulation of DNMT1.

biochemistry↗

bioRxiv2026

MISO: A Controlled Ablation of Masking, Initialization, Sampling, and Optimization for Segmentation in Volumetric Electron Microscopy

Multi-organelle segmentation in volumetric electron microscopy (vEM) faces several challenges, including severe class imbalance, the presence of small, rare classes, and inconsistent class coverage across crops. While recent work has focused primarily on architectural design, the impact of sampling, loss functions, and masking strategies on training effectiveness remains comparatively underexplored in vEM organelle segmentation. Here, we systematically evaluate sampling strategies, loss configurations, masking approaches, and model families (CNNs and vision transformers) on the CellMap benchmark. Using 289 annotated 3D FIB-SEM crops, we establish a 32-class segmentation benchmark with stratified train, validation, and test splits, and evaluate all the methods under the same training and inference settings. Across controlled ablations, the proposed combination of repeat-factor sampling, Tversky-BCE loss, and masking achieved the strongest rare-class performance, increasing rare-class mean Dice (mDice) from 0.3244 under uniform sampling to 0.3409. This corresponds to an absolute gain of +0.0165 mDice and a 5.1% relative improvement, while preserving comparable performance on common classes. Overall, we find that sampling, loss design, and masking contribute as much to performance variation as the choice of architecture, highlighting the importance of training-recipe design alongside model architecture in vEM organelle segmentation.

Cell Biology↗

bioRxiv2026

EZH2 Serine 21 Phosphorylation Restrains Compact-State PRC2 Activation and H3K27me3 Propagation

Polycomb Repressive Complex 2 (PRC2) propagates H3K27me3 through EED-dependent allosteric activation, yet how cells modulate the magnitude of this positive-feedback response remains poorly understood. Here, we identify phosphorylation of EZH2 serine 21 as a post-translational mechanism that attenuates PRC2 allosteric responsiveness. Prior structural studies have established that activator-bound PRC2 adopts both compact and extended active conformations. Using cryo-EM classification of wild-type and phospho-null EZH2 S21A PRC2 complexes, we find that the phospho-null EZH2 S21A substitution changes the distribution of particles across these pre-existing states, shifting PRC2 from a predominantly extended conformation to one enriched for the compact, allosterically activated conformation. Consistent with this structural transition, EZH2 S21A increases basal PRC2 activity, lowers the EC50 for H3K27me3-dependent stimulation, and accelerates H3K27me3 accumulation on peptide and nucleosome substrates. Disruption of the EED-EZH2 interface suppresses the S21A gain-of-activity phenotype, indicating that S21 phosphorylation constrains PRC2 by limiting productive EED-EZH2 allosteric coupling. In mesenchymal progenitor cells, loss of this phosphorylation-dependent restraint broadens H3K27me3 domains, reduces canonical PRC1 enrichment at high-occupancy Polycomb target loci, misregulates lineage-associated transcriptional programs, and impairs differentiation. These findings identify EZH2 S21 phosphorylation as a molecular rheostat that limits compact-state PRC2 activation, constrains H3K27me3 spreading, and preserves Polycomb-dependent developmental competence.

biochemistry↗

bioRxiv2026

Structure of the human HIRA histone chaperone with a nucleosome suggests a stepwise nucleosome assembly mechanism

The restoration of chromatin in the wake of a DNA or RNA polymerase is essential to maintain the integrity of eukaryotic genomes. Human HIRA is a 1.8-megadalton, three-subunit histone chaperone that mediates all replication-independent deposition of the histone variant H3.3 at active chromatin regions1-6. Disruption of HIRA perturbs active-chromatin organization and has wide-ranging consequences for development, cellular senescence, and genome integrity7-11. Despite its central biological role in reassembling nucleosomes post-transcription, the structure of native human HIRA and the mechanism by which it organizes histones and DNA during nucleosome assembly remain unknown. In particular, the function of the largest HIRA subunit CABIN1 is enigmatic. Here, we show that HIRA is not simply a passive histone hand-off factor but remains engaged across multiple stages of nucleosome assembly, including a close interaction with the nucleosome. Cryo-EM structures reveal that HIRA forms an extended arch-like structure that binds the nucleosome primarily through extensive CABIN1 contacts with histones, histone tails, nucleosomal DNA, and linker DNA, during the final stage of nucleosome assembly. Together, our results suggest a testable mechanism for HIRA-mediated nucleosome assembly and product release and provide the basis for elucidating the molecular details of this fundamental biological process.

biochemistry↗

bioRxiv2025

Structure-guided design and development of cyclic peptide allosteric activators of Polycomb Repressive Complex 2

Dysregulation of the histone methyltransferase Polycomb repressive complex 2 (PRC2) results in aberrant silencing of tumor suppressors and activation of oncogenes. Targeting PRC2 with compounds holds significant potential for both basic research and therapeutic applications. Here, we leveraged extensive structural studies of PRC2 to design a cyclic peptide that robustly activates PRC2. Structure-activity relationship studies guided the functional optimization of this cyclic peptide, yielding a Phenylalanine-type (Phe-type) cyclic peptide with approximately eight-fold activation compared to that of the poised state of PRC2. A 3.3[A] cryo-electron microscopy structure of the PRC2-peptide complex, combined with biochemical analyses, revealed a shift in the H3K27 methylation from mono-(me1) and dimethylation (me2) to trimethylation (me3). Finally, we demonstrated that the cyclic peptide exhibits improved mouse plasma stability and can also be readily taken up by cells which results in a shift of the H3K27 methylation landscape to trimethylation, similar to the observed effects in vitro. These findings support the utility of such molecules for probing PRC2 activation and targeting dysregulated H3K27 methylation in cancer.

biochemistry↗

bioRxiv2024

Diverse RNA Structures Induce PRC2 Dimerization and Inhibit Histone Methyltransferase Activity

Methyltransferase PRC2 (Polycomb Repressive Complex 2) deposits histone H3K27 trimethylation to establish and maintain epigenetic gene silencing. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications and, particularly, RNA. Research on PRC2-associated RNA has mostly focused on the tight-binding G-quadruplex (G4) RNAs, which inhibit PRC2 enzymatic activity in vitro and in cells, a mechanism explained by our recent cryo-EM structure showing G4 RNA-mediated PRC2 dimerization. However, PRC2 binds a wide variety of RNA sequences, and it remains unclear how diverse RNAs beyond G4 associate with and regulate PRC2. Here, we show that variations in RNA sequence elicit distinct effects on PRC2 function. A single-stranded G-rich RNA and an atypical G4 structure called a pUG-fold mediate PRC2 dimerization nearly identical to that induced by G4 RNA. In contrast, pyrimidine-rich RNAs, including a motif identified by CLIPseq in cells, do not induce PRC2 dimerization and instead bind PRC2 monomers with retention of methyltransferase activity. Only RNAs that dimerize PRC2 compete with nucleosome binding and inhibit PRC2 methyltransferase activity. CRISPR-dCas9 was adapted to localize different RNA elements onto a PRC2-targeted gene, revealing RNA sequence specificity for PRC2 regulation in cells. Thus, PRC2 binds many different RNAs with similar affinity, however, the functional effect on enzymatic activity depends entirely on the sequence of the bound RNA, a conclusion potentially applicable to any RNA- binding protein with a large transcriptome.

biochemistry↗

bioRxiv2024

Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator essential for embryonic development and maintenance of cell identity that trimethylates histone H3 at lysine 27 (H3K27me3) leading to gene silencing. PRC2 is regulated by association with protein cofactors and crosstalk with histone posttranslational modifications. Trimethylated histone H3 K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription where H3K27me3 is absent and inhibit PRC2 activity through unknown mechanisms. Using cryo-electron microscopy we reveal that histone H3 tails modified with H3K36me3 engage poorly with the PRC2 active site and preclude its effective interaction with chromatin, while the H3K4me3 modification binds to the allosteric site in the EED subunit, acting as an antagonist that competes with allosteric activators required for the spreading of the H3K27me3 repressive mark. Thus, the location along the H3 tail of the H3K4me3 and H3K36me3 modifications allow them to target two essential requirements for efficient trimethylation of histone H3K27. We further show that the JARID2 cofactor modulates PRC2 activity in the presence of these histone modifications.

biochemistry↗

bioRxiv2023

Structural basis for inactivation of PRC2 by G-quadruplex RNA

The histone methyltransferase PRC2 (Polycomb Repressive Complex 2) silences genes via successively attaching three methyl groups to lysine 27 of histone H3. PRC2 associates with numerous pre-mRNA and lncRNA transcripts with a binding preference for G-quadruplex RNA. Here, we present a 3.3[A]-resolution cryo-EM structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface comprised of two copies of the catalytic subunit EZH2, which limits nucleosome DNA interaction and occludes H3 tail accessibility to the active site. Our results reveal an unexpected mechanism for RNA-mediated inactivation of a chromatin-modifying enzyme. Furthermore, the flexible loop of EZH2 that helps stabilize RNA binding also facilitates the handoff between RNA and DNA, an activity implicated in PRC2 regulation by RNA. One-Sentence SummaryCryo-EM structure of RNA-bound PRC2 dimer elucidates an unexpected mechanism of PRC2 inhibition by RNA.

biochemistry↗

bioRxiv2022

DNMT1 inhibition by pUG-fold quadruplex RNA

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a non-canonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicates that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

biochemistry↗

bioRxiv2020

JARID2 and AEBP2 regulate PRC2 activity in the presence of H2A ubiquitination or other histone modifications

The Polycomb repressive complexes PRC1 and PRC2 functionally interact to coordinate cell type identity by the epigenetic regulation of gene expression. It has been proposed that PRC2 is recruited to genomic loci via the recognition of PRC1-mediated mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub1), but the mechanism of this process remains poorly understood. Here, we report the cryo-EM structure of human PRC2 with cofactors JARID2 and AEBP2 bound to a nucleosome substrate containing H2AK119ub1. We find that JARID2 and AEBP2 each interact with one of the two ubiquitin molecules in the nucleosome. A ubiquitin-interaction motif (UIM) in JARID2 is sandwiched between ubiquitin and the histone H2A-H2B acidic patch. Simultaneously, the tandem zinc-fingers of AEBP2 interact with the second ubiquitin and the histone H2A-H2B surface on the opposite side of the nucleosome. JARID2 plays a dual role in the H2AK119ub1 dependent stimulation of PRC2 through interactions with both EED via its K116 trimethylation and with the H2AK119-ubiquitin. AEBP2, on the other hand, appears to primarily serve as a scaffold contributing to the interaction between PRC2 and the H2AK119ub1 nucleosome. Our structure also provides a detailed visualization of the EZH2-nucleosome interface, revealing a segment of EZH2 (named "bridge helix") that is stabilized as it bridges the EZH2(SET) domain, the H3 tail and the nucleosomal DNA. In addition to the role played by AEBP2 and JARID2 in PRC2 regulation by H2AK119ub1 recognition, we also observe that the presence of these cofactors partially overcomes the inhibitory effect that H3K4- and H3K36-trimethylation have on core PRC2. Together, our results reveal the central role played by cofactors JARID2 and AEBP2 in orchestrating the crosstalk between histone post-translational modifications and PRC2 methyltransferase activity.

biophysics↗

bioRxiv2018

Visualization of PRC2-Dinucleosome Interactions Leading to Epigenetic Repression

Epigenetic regulation is mediated by protein complexes that couple recognition of chromatin marks to activity or recruitment of chromatin-modifying enzymes. Polycomb repressive complex 2 (PRC2), a gene silencer that methylates lysine 27 of histone H3, is stimulated upon recognition of its own catalytic product, and has been shown to be more active on dinucleosomes than H3 tails or single nucleosomes. These properties likely facilitate local H3K27me2/3 spreading causing heterochromatin formation and gene repression. Here, cryo-EM reconstructions of human PRC2 bound to dinucleosomes show how a single PRC2, interacting with nucleosomal DNA, precisely positions the H3 tails to recognize a H3K27me3 mark in one nucleosome and is stimulated to modify a neighboring nucleosome. The geometry of the PRC2-DNA interactions allow PRC2 to tolerate different dinucleosome geometries due to varying lengths of the linker DNA. Our structures are the first to illustrate how an epigenetic regulator engages with a complex chromatin substrate.

biophysics↗