Biology subjects

Kanemaki, M. T.

Publications and source records attributed to Kanemaki, M. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Firrm functions as a multiprotein interaction platform that coordinates Mcm8-9-Hrob recruitment and Fignl1-mediated Rad51 unloading

Rad51 forms nucleoprotein filaments to catalyze homology search and strand exchange. The transition to recombination-associated DNA synthesis is accompanied by Rad51 unloading and recombination helicase recruitment. However, how these reactions are coordinated remains unclear. Using Xenopus egg extracts, we identify a mechanistic coupling between Rad51 unloading and recombination helicase recruitment. Proteomic analysis revealed that Rad51 filaments assembled on double-stranded DNA recruit a limited set of recombination regulators, including C1orf112/Firrm, Fignl1, Mcm8, Mcm9, and Hrob. Firrm forms a stoichiometric complex with Fignl1 and directly binds short acidic motifs in the intrinsically disordered regions of both Mcm9 and Hrob. The Firrm-Mcm9 interaction promotes recruitment of Mcm8-9 and Hrob to Rad51 filaments. Purified Mcm8-9 stimulates the ATPase activity of Fignl1 through Firrm in a DNA-dependent manner. Rad51 unloading from double-stranded DNA depends primarily on Fignl1 ATPase activity and is stimulated by Firrm-Mcm9 interaction. In human cells, FIRRM-FIGNL1 and MCM8-9-HROB act in the same genetic pathway to confer resistance to DNA interstrand crosslinks. These results support a model in which Rad51 filaments and Firrm-Fignl1 promote recruitment of Mcm8-9-Hrob, which, in turn, stimulates Fignl1 ATPase activity, thereby coupling recombination helicase recruitment with Rad51 unloading.

molecular biology↗

bioRxiv2026

Cohesin residence time gates 3D genome response to histone hyperacetylation

Cohesin-mediated loop extrusion and chromatin state-dependent compartmentalization are major drivers of three-dimensional (3D) genome organization. Although epigenomic perturbations are widely assumed to reshape chromatin architecture, the mechanisms that determine how changes in chromatin state are translated into structural reorganization remain poorly understood. Here, we identify cohesin residence time as a key regulator of the genomes architectural response to histone hyperacetylation induced by histone deacetylase inhibition (HDACi). Acute depletion of RAD21 or CTCF weakens chromatin loops but preserves HDACi-induced changes in compartmentalization, contact-scaling behavior, and loop density. In contrast, perturbation of cohesin loading or release produces opposing effects: NIPBL depletion sensitizes and amplifies architectural responses to HDACi, whereas WAPL loss renders the genome largely refractory to HDACi-induced remodeling, suppressing changes in compartments and loop density while stabilizing CTCF-anchored loops. These distinct architectural outcomes occur despite comparable levels of HDACi-induced histone hyperacetylation across genotypes, indicating that differential epigenomic input is not responsible for the observed effects. Together, our findings demonstrate that dynamic cohesin turnover, rather than cohesin chromatin association alone, governs whether epigenomic perturbations are converted into higher-order genome reorganization. These results establish cohesin residence time as a molecular gate linking chromatin state to 3D genome architecture and reveal a previously unrecognized principle underlying chromatin architecture plasticity.

genomics↗

bioRxiv2026

m1A58 governs two steps of human initiator-methionine tRNA metabolism: maturation and XRN2-mediated decay

N{superscript 1}-methyladenosine at position 58 (m{superscript 1}A58), installed by the TRMT6/TRMT61A complex, is a highly conserved structural modification implicated in tRNA stability and human disease. In yeast, loss of m{superscript 1}A58 disrupts initiator methionine tRNA (tRNA) metabolism through defects in precursor processing and rapid tRNA decay, but whether these mechanisms operate in human cells has remained unclear. Here, using acute degron-mediated depletion of TRMT6 in human HCT116 cells combined with precursor-resolved tRNA sequencing, we show that m{superscript 1}A58 controls two distinct steps of tRNA metabolism. TRMT6 depletion selectively reduces mature tRNA while causing accumulation of precursor species retaining both 5'-leader and 3'-trailer sequences, consistent with impaired maturation. In parallel, the nuclear 5'[->]3' exonuclease XRN2 selectively degrades the residual mature hypomodified tRNA pool without affecting precursor accumulation. Loss of mature tRNA activates the integrated stress response and impairs proliferation, which is restored by XRN2 co-depletion. Together, our findings identify m{superscript 1}A58 as a coordinator of human initiator-tRNA maturation and surveillance, establishing a two-step pathway that maintains tRNA homeostasis in human cells.

biochemistry↗

bioRxiv2025

The mammalian rapid tRNA decay pathway is critical for N⁷-methylguanosine-hypomodified tRNA degradation under physiological conditions

Chemical modifications of transfer RNAs (tRNAs) are integral to their stability and to translation. Loss of N7-methylguanosine (m7G) on specific tRNAs reduces their steady-state abundance and impairs translation in mammals, but whether these decreases reflect active degradation under physiological growth conditions is unresolved. Here, using human HCT116 cells, we show that knockdown of the 5'[->]3' exonuclease, XRN2, restores tRNA levels diminished by METTL1 depletion. Leveraging conditional protein knockdown, we performed time-resolved measurements of mature tRNA levels and directly quantified decay kinetics. We show that in the absence of heat stress, mG-hypomodified tRNAs undergo XRN2-dependent accelerated decay. Finally, partial loss of the Drosophila XRN2 ortholog, Rat1, genetically rescues male sterility of mettl1 mutants, demonstrating organismal relevance. These findings define a conserved constitutive rapid tRNA decay pathway in mammals and indicate inhibition of tRNA decay as a potential therapeutic strategy in disorders caused by tRNA hypomodification.

molecular biology↗

bioRxiv2025

Cohesin acts as a transcriptional gatekeeper by restraining pause-release to promote processive elongation

Cohesin organizes 3D chromatin architecture, including promoter-enhancer loops, yet its loss has surprisingly modest effects on steady-state gene expression. We address this paradox by demonstrating that cohesin acts at different stages of transcription. First, it promotes Pol II promoter recruitment by facilitating enhancer-promoter communication that maintains active promoter chromatin states. Second, it delays pause release by transiently associating with the transcriptional machinery during the pause-release transition. Kinetic modelling suggests that reduced Pol II recruitment and enhanced pause release have compensatory effects, contributing to minimal changes in steady-state gene expression across genes upon cohesin loss. In contrast, cohesin depletion impairs robust transcriptional induction in response to external stimuli. Moreover, cohesin ensures sufficient pausing duration as a quality-control-like step to promote elongation complex assembly and transcription processivity. Here, we show that cohesin regulates multiple steps of transcription, offering mechanistic insight into cohesin-related diseases, including cancers and cohesinopathies.

molecular biology↗

bioRxiv2025

Cohesin drives chromatin scanning during the RAD51-mediated homology search.

Cohesin folds genomes into chromatin loops, whose roles are under debate. We report that double strand breaks (DSB) induce de novo formation of chromatin loops, with the break positioned at the loop base. These loops form only in S/G2 phases and occur during repair via homologous recombination (HR), concomitant with DNA end resection and RAD51 assembly. RAD51 showed two-tiered accumulation around DSBs, with a broad (~Mb) domain arising from the homology search. This domain is regulated by cohesin unloader, is constrained by TAD boundaries, and it overlaps with chromatin regions reeled through the break-anchored loop, suggesting that loop extrusion regulates the homology search. Indeed, depletion of NIPBL results in reduced HR, and this effect is more pronounced when the HR donor is far (~100 kb) from the break. Our data indicates that loop-extruding cohesin promotes the mammalian homology search by facilitating break-chromatin interactions within the damaged TAD. One-Sentence SummaryHigh spatiotemporal resolution analysis of double strand beak repair in 3D genome revealed the role of cohesin-driven loop extrusion in the homology search.

cell biology↗

bioRxiv2025

A single-chain antibody-based AID2 system for conditional degradation of GFP-tagged and untagged proteins

Protein knockdown using an improved auxin-inducible degron (AID2) technology has proven to be a powerful tool for studying protein function. The current approach requires the fusion of target proteins with a degron tag, a process typically achieved through CRISPR knock-in. However, knock-in remains challenging in non-model organisms and humans, limiting the broader applicability of AID2. To overcome this limitation, we developed a single-chain antibody AID2 (scAb-AID2) system. This approach employs an adaptor composed of a single-chain antibody fused with a degron, which recognises a target protein and induces rapid degradation in the presence of the inducer 5-Ph-IAA. We demonstrated that scAb-AID2, in combination with an anti-GFP nanobody, degraded GFP-fused proteins in human cells and C. elegans. Furthermore, we showed that endogenous p53 and H/K-RAS were conditionally degraded in cells expressing an adaptor encoding an anti-p53 nanobody and -RAS monobody, respectively, and led to aphidicolin sensitivity in cell culture and growth inhibition in mouse xenografts. This study paves the way for broader application of AID2-based target depletion in model and non-model organisms and for advancing therapeutic strategies.

bioengineering↗

bioRxiv2024

Nuclear blebs are associated with destabilized chromatin packing domains

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e., herniations of the nuclear envelope) have been induced by (1) nuclear compression, (2) nuclear migration (e.g., cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated if (1) packing domain organization is altered within nuclear blebs and (2) if alteration in packing domain structure contributed to bleb formation. Using Dual-Partial Wave Spectroscopic microscopy, we show that chromatin packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that translocation into blebs results in a fragmented higher-order chromatin structure. SUMMARY STATEMENTNuclear blebs are linked to various pathologies, including cancer and premature aging disorders. We investigate alterations in higher-order chromatin structure within blebs, revealing fragmentation of nanoscopic heterochromatin domains.

bioengineering↗

bioRxiv2024

Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins and enhance target depletion. For this purpose, we initially compared PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrated control of cohesin and condensin with AID2 and BromoTag, respectively. We developed a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrated that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we found that co-depletion of ORC1 and CDC6 by the double-degron system completely suppressed DNA replication, and the cells entered mitosis with single-chromatid chromosomes, indicating DNA replication was uncoupled from the cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

cell biology↗

bioRxiv2024

Cohesin regulates promoter-proximal pausing of RNA Polymerase II by limiting recruitment of super elongation complex

Cohesin is a ring-shaped complex, responsible for establishing sister chromatid cohesion and forming topologically associating domains (TADs) and chromatin loops. Loss-of-function mutations in cohesin subunits and its regulatory factors can cause Cornelia de Lange syndrome (CdLS). Because dysregulated gene expression was observed in CdLS, it has long been thought that cohesin plays a regulatory role in transcription. Here, we investigated the effect of acute cohesin depletion on transcription and observed that a small number of genes exhibited differential expression. Analysis of RNA polymerase II (Pol II) distribution revealed that the depletion reduced Pol II promoter binding and pausing simultaneously at the majority of genes. This implies that at most genes, the two decreases counterbalance each other, resulting in unchanged gene expression. Additionally, we find that cohesin loss increased promoter binding of super elongation complex (SEC), which mediates the release of Pol II from paused state. Moreover, the reduction in pausing caused by cohesin depletion was no longer observed when SEC was inhibited. These observations suggest that cohesin regulates Pol II pausing by restricting SEC recruitment to promoters. Together, our study demonstrates the involvement of cohesin in transcriptional regulation, particularly in Pol II pause and release.

molecular biology↗

bioRxiv2023

Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

molecular biology↗

bioRxiv2023

Depletion of lamins B1 and B2 alters chromatin mobility and induces differential gene expression by a mesoscale-motion dependent mechanism

BACKGROUNDB-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology. RESULTSPaired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, in situ Hi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning. CONCLUSIONSOur findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.

bioengineering↗

bioRxiv2023

TRAIP plays an essential role in the dissociation of PCNA from chromatin during mitosis

Loading PCNA onto chromatin is a pivotal step in DNA replication, cell cycle progression, and genome integrity. Conversely, unloading PCNA from chromatin is equally crucial for maintaining genome stability. Cells deficient in the PCNA unloader ATAD5-RFC exhibit elevated levels of chromatin-bound PCNA during S phase, but still show dissociation of PCNA from chromatin in mitosis. In this study, we found that depletion of TRAIP, an E3 ubiquitin ligase, results in the retention of PCNA on chromatin during mitosis. Although TRAIP-depleted cells with chromatin-bound PCNA during mitosis progressed into the subsequent G1 phase, they displayed reduced levels of Cdt1, a key replication licensing factor, and impaired S phase entry. In addition, TRAIP-depleted cells exhibited delayed S phase progression. These results suggest that TRAIP functions independently of ATAD5-RFC in removing PCNA from chromatin. Furthermore, TRAIP appears to be essential for precise pre-replication complexes (pre-RCs) formation necessary for faithful initiation of DNA replication and S phase progression.

molecular biology↗

bioRxiv2022

Ubiquitin ligase TRAIP plays an essential role during the S-phase of unperturbed cell cycle in the resolution of DNA replication - transcription conflicts

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, for the first time, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We find that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.

cell biology↗

bioRxiv2022

MCMBP promotes the assembly of the MCM2-7 hetero-hexamer to ensure robust DNA replication in human cells

The MCM2-7 hetero-hexamer is the replicative DNA helicase that plays a central role in eukaryotic DNA replication. In proliferating cells, the expression level of the MCM2-7 hexamer is kept high, which safeguards the integrity of the genome. However, how the MCM2-7 hexamer is assembled in living cells remains unknown. Here, we revealed that the MCM-binding protein (MCMBP) plays a critical role in the assembly of this hexamer in human cells. MCMBP associates with MCM3 which is essential for maintaining the level of the MCM2-7 hexamer. Acute depletion of MCMBP demonstrated that it contributes to MCM2-7 assembly using nascent MCM3. Cells depleted of MCMBP gradually ceased to proliferate because of reduced replication licensing. Under this condition, p53-positive cells exhibited arrest in the G1 phase, whereas p53-null cells entered the S phase and lost their viability because of the accumulation of DNA damage, suggesting that MCMBP is a potential target for killing p53-deficient cancers.

molecular biology↗

bioRxiv2021

Global landscape of replicative DNA polymerase usage in the human genome

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Pol{varepsilon} and Pol usage genome wide. The polymerase usage profiles show Pol{varepsilon} synthesises the leading strand and Pol contributes mainly to lagging strand synthesis. Combining the Pol{varepsilon} and Pol profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co- directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

molecular biology↗

bioRxiv2021

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

cell biology↗

bioRxiv2019

The RIF1-Long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

molecular biology↗