Biology subjects

Narlikar, G. J.

Publications and source records attributed to Narlikar, G. J..

11 recordsLinked to original sources

bioRxiv2026

ATP-dependent reorientation on a nucleosome primes INO80 for DNA translocation

ATP-dependent chromatin remodeling enzymes play central roles in genome regulation by mobilizing nucleosomes. All remodelers except for one, position their core ATPase at an internal nucleosome location, superhelical location (SHL) 2, and translocate DNA from this site to alter nucleosome conformation. The exception, the remodeler INO80, positions its core ATPase, Ino80, near the DNA exit/entry site, SHL -6. This major architectural difference has raised the question of whether INO80 acts by a fundamentally different mechanism from other remodelers. Here, using cryogenic electron microscopy we capture a new conformation of INO80 that is substantially enriched upon ATP hydrolysis and has Ino80 positioned at an internal nucleosomal location. Comprehensive conformational landscape analysis further uncovers an ATP hydrolysis dependent continuum of additional INO80 conformational states on a nucleosome that were not previously detected. Our studies provide compelling evidence to support a model where INO80 initially engages nucleosomes near SHL -6, followed by a dramatic ATP hydrolysis dependent ~180{degrees} reorientation around the nucleosome to place its Ino80 near SHL -2 from where DNA is translocated. INO80s unique reorientation has broad implications for understanding how ATP-dependent steps that precede nucleosome mobilization can increase the fidelity of remodeling by being responsive to nucleosomal cues.

biophysics↗

bioRxiv2026

Preferential formation of NUP98-KDM5A condensates at specific H3K4me3-rich loci drives leukemogenic gene expression

Chromosomal translocations involving NUP98 generate fusion proteins that alter gene expression programs, yet the fundamental principles governing their gene targeting and condensate behavior remain poorly understood. Using NUP98-KDM5A as a model, we integrate cellular imaging, in vitro reconstitution, and genomic analyses to dissect how chromatin engagement shapes condensate formation. We find that NUP98-KDM5A forms sub-diffraction-limited, gel-like condensates whose assembly is potentiated by binding to H3K4me3. This interaction creates a quantitative targeting mechanism in which, at the native expression level, condensates preferentially form at genomic loci with high local H3K4me3 density. Such local density-dependent recruitment explains selective enrichment at the leukemogenic HOX gene clusters, despite widespread presence of H3K4me3 across the genome. Analysis of single-cell sequencing data from patients further supports a correlation between local H3K4me3 density and transcriptional activation in NUP98-KDM5A-driven leukemia. Together, our findings reveal how activating chromatin marks and condensate-forming proteins synergize to generate specificity within euchromatin, offering a generalizable framework for understanding how chromatin-associated condensates interpret epigenetic landscapes.

cell biology↗

bioRxiv2024

Chromatin mimicry by human JC virus

Chronically persistent viruses are integral components of the organismal ecosystem in humans and animals 1 2. Many of these viruses replicate and accumulate within the cell nucleus 3. The nuclear location allows viruses to evade cytoplasmic host viral sensors and promotes viral replication 4. One of the unexplored and puzzling aspects of the viral nuclear lifecycle involves the viruss ability to deal with the physical constraints of nuclear architecture. To replicate and accumulate within the nucleus in large numbers sufficient for infection spreading, DNA viruses need to overcome the spatial limitations imposed by chromatin and the nuclear matrix. We found that one of the most widespread and potentially lethal human viruses, the JC polyomavirus 5, interferes with nuclear heterochromatin to create virus-occupied space. The JC viruss impact on heterochromatin is mediated by the viral nonstructural protein, Agnoprotein (Agno). Agnos interference with heterochromatin is governed by structurally diverse mimics of host epigenetic regulators that facilitate virus-induced chromatin reorganization and a dramatic decline in nuclear stiffness in the infected cells. The JCV epigenetic mimicry is critical for the virus infection, as evident from reduced replication of mimic-mutant viruses. Our data suggest that modulation of nuclear mechanical properties is a novel strategy enabling chronicity of the JC and possibly other nuclear virus infections.

microbiology↗

bioRxiv2023

Understanding how genetically encoded tags affect phase separation by Heterochromatin Protein HP1α

Liquid-liquid phase separation (LLPS) is driven by weak multi-valent interactions. Such interactions can result in the formation of puncta in cells and droplets in vitro. The heterochromatin protein HP1 forms droplets with chromatin in vitro and is found in puncta in cells. A common approach to visualize the dynamics of HP1 in cells is to genetically encode fluorescent tags on the protein. HP1 modified with tags such as GFP has been shown to localize to dynamic puncta in vivo. However, whether tagged HP1 retains its intrinsic phase separation properties has not been systematically studied. Here, using different C-terminal tags (AID-sfGFP, mEGFP, and UnaG), we assessed how tag size and linker length affected the phase separation ability of HP1 with DNA in vitro. We found that the AID-sfGFP tag (52 kDa) promoted HP1 phase-separation, possibly driven by the highly disordered AID degron. The mEGFP tag (27 kDa) inhibited phase-separation by HP1, whereas an UnaG tag (13 kDa) with a 16 amino acid linker showed minimal perturbation. The UnaG tag can thus be used in cellular studies of HP1 to better correlate in vitro and in vivo studies. To test if cellular crowding overcomes the negative effects of large tags in vivo, we used polyethylene glycol (PEG) to mimic crowding in vitro. We found that addition of 10% PEG8000 or PEG4000 enables phase separation by GFP-tagged HP1 at comparable concentrations as untagged HP1. However, these crowding agents also substantially dampened the differences in phase-separation between wild-type and mutant HP1 proteins. PEG further drove phase-separation of Maltose Binding Protein (MBP), a tag often used to solubilize other proteins. These results suggest that phase-separation of biological macromolecules with PEG should be interpreted with caution as PEG-based crowding agents may change the types of interactions made within the phases.

biophysics↗

bioRxiv2021

Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

ATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.

genomics↗

bioRxiv2020

HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

In mammals HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1, HP1{beta}, and HP1{gamma}, display rapid on-off dynamics. Here we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1-DNA condensates, HP1 acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1{beta}. Finally, we find that differences in each HP1 paralogs DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

biochemistry↗

bioRxiv2020

Massively multiplex single-molecule oligonucleosome footprinting

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular states of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and visualizing higher-order chromatin structure. 1-sentence summaryHigh-throughput single-molecule real-time footprinting of chromatin arrays reveals heterogeneous patterns of oligonucleosome occupancy.

genomics↗

bioRxiv2020

ATP hydrolysis by the SNF2 domain of the ultraspecific maintenance methylase Dnmt5 drives recognition and modification of hemimethylated DNA

C. neoformans Dnmt5 is an ultraspecific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step that is essential for DNA methylation. Such coupling occurs independently of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating the Dnmt5 ATP binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a flipped-out conformation of the target cytosine bypass the SNF2 domains requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to base-flipping by the DNMT domain. These findings establish a new role for a SNF2 ATPase domain: controlling substrate recognition and catalysis by an adjoined enzymatic domain. This coupling may contribute to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.

molecular biology↗

bioRxiv2019

Electron cryo-microscopy structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, are disordered. The disordered acidic patch is expected to inhibit the second SNF2h promoter, while disorder near the dyad is expected to promote DNA translocation. The other structure doesnt show octamer deformation, but surprisingly shows a 2bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation. One sentence summaryCryo-EM structures capture different conformational states of chromatin remodeler-nucleosome complexes.

biophysics↗

bioRxiv2018

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets

The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

biophysics↗

bioRxiv2017

Epigenetic maintenance of DNA methylation after evolutionary loss of the de novo methyltransferase

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles, yet has been lost many times in diverse eukaryotic lineages. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase, Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 MYA. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 MY through a process analogous to Darwinian evolution of the genome.

molecular biology↗