Biology subjects

Galonic Fujimori, D.

Publications and source records attributed to Galonic Fujimori, D..

6 recordsLinked to original sources

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↗

bioRxiv2025

Structural analysis of 23S rRNA methylating enzyme Cfr reveals RNA binding determinants for methylation regiospecificity and antibiotic resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPSA phenotype). Cfr catalyses methylation of the C8 position of the A2503 base in 23S rRNA; the recognition site of the above antibiotic classes. Along with RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfrs dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb the PhLOPSA resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in-cellulo 23S rRNA methylated species, and in-cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multi-drug resistance activity. In particular, we found that Cfrs Q329 residue is important for C8-specific methylation. These data provide a framework for further studies of the biochemistry, structure and inhibition of this important resistance determinant.

microbiology↗

bioRxiv2024

Histone demethylase enzymes KDM5A and KDM5B modulate immune response by suppressing transcription of endogenous retroviral elements

Epigenetic factors, including lysine-specific demethylases such as the KDM5 paralogs KDM5A and KDM5B have been implicated in cancer and the regulation of immune responses. Here, we performed a comprehensive multiomic study in cells lacking KDM5A or KDM5B to map changes in transcriptional regulation and chromatin organization. RNA-seq analysis revealed a significant decrease in the expression of Kruppel-associated box containing zinc finger (KRAB-ZNF) genes in KDM5A or KDM5B knockout cell lines, which was accompanied by changes ATAC-seq and H3K4me3 ChIP-seq. Pharmacological inhibition of KDM5A and KDM5B catalytic activity with a pan-KDM5 inhibitor, CPI-455, did not significantly change KRAB-ZNF expression, raising the possibility that regulation of KRAB-ZNF expression does not require KDM5A and KDM5B demethylase activity. KRAB-ZNF are recognized suppressors of the transcription of endogenous retroviruses (ERVs) and HAP1 cells with KDM5A or KDM5B gene inactivation showed elevated ERV expression, increased dsRNA levels and elevated levels of immune response genes. Acute degradation of KDM5A using a dTAG system in HAP1 cells led to increased ERV expression, demonstrating that de-repression of ERV genes occurs rapidly after loss of KDM5A. Co-immunoprecipitation of KDM5A revealed an interaction with the Nucleosome Remodeling and Deacetylase (NuRD) complex suggesting that KDM5A and NuRD may act together to regulate the expression of ERVs through KRAB-ZNFs. These findings reveal roles of KDM5A and KDM5B in modulating ERV expression and underscore the therapeutic potential of using degraders of KDM5A and KDM5B to modulate tumor immune responses. Author SummaryThe histone demethylases KDM5A and KDM5B are transcriptional repressors that play an important role in cancer and immune response, making them attractive drug targets. Unfortunately, small molecule inhibitors, including CPI-455, that block KDM5A and KDM5B enzymatic activity, have shown only limited effectiveness at suppressing cancer cell viability as single agents in vitro. In this study we undertook a multi-omics approach to map transcriptional and chromatin changes in KDM5A and KDM5B deficient cells compared to those treated with CPI-455. The datasets revealed that KDM5A and KDM5B modulate the expression of KRAB-ZNF genes and that loss of either gene was associated with increased expression of ERV genes and upregulation of immune response markers. Surprisingly, pharmacological inhibition of these enzymes did not phenocopy genetic ablation. In contrast, acute degradation of KDM5A using a dTAG system caused an increase in ERV expression, providing evidence that this immune modulation is independent of demethylase activity. Together with the limited success of small molecule inhibitors, our data provide strong rationale for the development of KDM5A and KDM5B degraders to modulate tumor immune responses.

genomics↗

bioRxiv2022

X-linked intellectual disability mutations alter the conformational state of a histone demethylase's sensing of chromatin

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie between the segments of the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. Through in vitro binding and kinetic studies using nucleosomes, we find that while the ARID domain is required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. In addition, the unstructured linker region between the ARID and PHD1 domains interacts with PHD1 and is necessary for nucleosome binding. Our data suggests a model in which the PHD1 domain inhibits DNA recognition by KDM5C. This inhibitory effect is relieved by the H3 tail, enabling recognition of flanking DNA on the nucleosome. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains break this regulation by enhancing DNA binding, resulting in the loss of specificity of substrate chromatin recognition and rendering demethylase activity lower in the presence of flanking DNA. Our findings suggest a model by which specific XLID mutations could alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.

biochemistry↗

bioRxiv2021

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.

biophysics↗

bioRxiv2021

Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant which achieves near-stoichiometric methylation of rRNA, we determined a 2.2 [A] cryo-EM structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

biochemistry↗