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A structure-guided small-molecule and chemoproteomics approach uncovers a catalytic site inhibitor selective for the jumonji subfamily of H3K27me3 demethylases; the inhibitor decreases lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages. JMJ demethylase inhibitors and inflammation Jumonji (JMJ) family histone demethylases have become recognized as key regulators of transcription, although the importance of their enzymatic activity - as opposed to their structural role - has been unclear. JMJD3 demethylases are specific for histone H3 trimethylated at Lys27 (H3K27me3) and are involved in the inflammatory response, as well as other physiological functions. Here, a structure-guided small-molecule and chemoproteomics approach is used to discover a catalytic-site inhibitor selective for the H3K27me3-specific JMJ subfamily. The inhibitor reduces lipopolysaccharide-induced pro-inflammatory cytokine production in human primary macrophages. This study demonstrates the importance of JMJ demethylase activity and suggests that small-molecule inhibitors of JMJ enzymes could have therapeutic applications. The jumonji (JMJ) family of histone demethylases are Fe 2+ - and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes 1 , 2 , 3 , 4 . These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context 5 . Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance 2 , 4 , 6 , as well as in development, physiology and disease 3 , 7 . However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX) 8 . The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Methods for quantifying gene expression 1 and chromatin accessibility 2 in single cells are well established, but single-cell analysis of chromatin regions with specific histone modifications has been technically challenging. In this study, we adapted the CUT&Tag method 3 to scalable nanowell and droplet-based single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues and during the differentiation of human embryonic stem cells. We focused on profiling polycomb group (PcG) silenced regions marked by histone H3 Lys27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we used scCUT&Tag to profile H3K27me3 in a patient with a brain tumor before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment. An improved method for single-cell analysis of histone modifications is applied to stem cell differentiation and cancer.

Cancer is a multifaceted disease driven by genetic mutations and epigenetic dysregulation. Among epigenetic modifiers, histone demethylases like KDM4C (lysine demethylase 4C) play a pivotal role in tumor progression by removing repressive methylation mark at Histone H3K9/H3K36 and altering chromatin structure and gene expression. Overexpression of KDM4C has been implicated in various malignancies, including breast, prostate, colorectal, and hepatocellular carcinomas, hence it is promising drug target. This study employs a structure-based drug discovery strategy to identify natural polyphenolic inhibitors of KDM4C. High-throughput virtual screening, followed by molecular docking, molecular dynamics (MD) simulations, and MM-GBSA free energy calculations, used to assess binding potential. Pectolinarin and compound 202 emerged as top candidates, outperforming the reference ligand (6X9) used from PDBID: 5KR7, in docking scores, and exhibiting robust hydrogen bonding and hydrophobic interactions within the active site. MD simulations over 200 ns confirmed complex stability, indicated by consistently low RMSD and RMSF values. MM-GBSA analysis revealed strong binding affinities with free energy values of −68.4 kcal/mol and −65.7 kcal/mol for Pectolinarin and compound 202, respectively. ADMET predictions supported their drug-likeness, suggesting favorable pharmacokinetic profiles, oral bioavailability, and low toxicity. These findings highlight pectolinarin and compound 202 as promising leads for KDM4C-targeted cancer therapy. Further experimental validation is required to confirm their efficacy and specificity. Overall, this work demonstrates the potential of computational approaches in advancing the discovery of nature-derived epigenetic therapeutics.

Floral transition from the vegetative to the reproductive growth phase is a major change in the plant life cycle and a key factor in reproductive success. In rice (Oryza sativa L.), a facultative short-day plant, numerous flowering time and flower formation genes that control floral transition have been identified and their physiological effects and biochemical functions have been clarified. In the present study, we used a Se14-deficient mutant line (HS112) and other flowering mutant lines to investigate the photoperiodic response, chromosomal location and function in the photoperiod sensitivity of the Se14 gene. We also studied the interactive effects of this locus with other crucial flowering time genes. We found that Se14 is independent of the known photoperiod-sensitive genes, such as Hd1 and Ghd7, and is identical to Os03g0151300, which encodes a Jumonji C (JmjC) domain-containing protein. Expression analysis revealed that the expressions of RFT1, a floral initiator known as a \"florigen-like gene\", and Ehd1 were up-regulated in HS112, whereas this up-regulation was not observed in the original variety of 'Gimbozu'. ChIP assays of the methylation states of histone H3 at lysine 4 (H3K4) revealed that the trimethylated H3K4 in the promoter region of the RFT1 chromatin was significantly increased in HS112. We conclude that Se14 is a novel photoperiod-sensitivity gene that has a suppressive effect on floral transition (flowering time) under long day-length conditions through the modification of chromatin structure by H3K4me3 demethylation in the promoter region of RFT1.

Background SET domain-containing histone lysine methyltransferases (HKMTs) and JmjC domain-containing histone demethylases (JHDMs) are essential for maintaining dynamic changes in histone methylation across parasite development and infection. However, information on the HKMTs and JHDMs in human pathogenic piroplasms, such as Babesia duncani and Babesia microti , and in veterinary important pathogens, including Babesia bigemina , Babesia bovis , Theileria annulata and Theileria parva , is limited. Results A total of 38 putative KMTs and eight JHDMs were identified using a comparative genomics approach. Phylogenetic analysis revealed that the putative KMTs can be divided into eight subgroups, while the JHDMs belong to the JARID subfamily, except for BdJmjC1 (BdWA1_000016) and TpJmjC1 (Tp Muguga_02g00471) which cluster with JmjC domain only subfamily members. The motifs of SET and JmjC domains are highly conserved among piroplasm species. Interspecies collinearity analysis provided insight into the evolutionary duplication events of some SET domain and JmjC domain gene families. Moreover, relative gene expression analysis by RT‒qPCR demonstrated that the putative KMT and JHDM gene families were differentially expressed in different intraerythrocytic developmental stages of B. duncani , suggesting their role in Apicomplexa parasite development. Conclusions Our study provides a theoretical foundation and guidance for understanding the basic characteristics of several important piroplasm KMT and JHDM families and their biological roles in parasite differentiation.

Although the DNA damage–induced ubiquitylation by RNF8 and/or RNF168 ubiquitin ligases is crucial for the DNA-damage response (DDR), the precise mechanisms of ubiquitylation-mediated chromatin modulation and recruitment of DNA-repair proteins 53BP1 and RAP80–BRCA1 are not fully understood. A new study now indicates that human demethylase JMJD1C regulates the RAP80–BRCA1 branch of this DDR pathway. Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80–BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80–BRCA1 branch of this DNA-damage response (DDR) pathway. JMJD1C was stabilized by interaction with RNF8, was recruited to DSBs, and was required for local ubiquitylations and recruitment of RAP80–BRCA1 but not 53BP1. JMJD1C bound to RNF8 and MDC1, and demethylated MDC1 at Lys45, thereby promoting MDC1-RNF8 interaction, RNF8-dependent MDC1 ubiquitylation and recruitment of RAP80–BRCA1 to polyubiquitylated MDC1. Furthermore, JMJD1C restricted formation of RAD51 repair foci, and JMJD1C depletion caused resistance to ionizing radiation and PARP inhibitors, phenotypes relevant to aberrant loss of JMJD1C in subsets of breast carcinomas. These findings identify JMJD1C as a DDR component, with implications for genome-integrity maintenance, tumorigenesis and cancer treatment.

Constitutive heterochromatin (HC) is important for maintaining chromosome stability, but also delays the repair of DNA double-strand breaks (DSBs). DSB repair in complex mammalian genomes involves a fast phase (2–6 h) in which most of the breaks are rapidly repaired, and a slow phase (up to 24 h) in which the remaining damages in HC are repaired. We found that p53 deficiency delays the slow-phase DNA repair after ionizing irradiation. p53 deficiency prevents downregulation of histone H3K9 trimethylation at the pericentric HC after DNA damage. Moreover, p53 directly induces expression of the H3K9 demethylase Jumonji domain 2 family demethylase (JMJD2b) through promoter binding. The p53 activation also indirectly downregulates expression of the H3K9 methyltransferase SUV39H1. Depletion of JMJD2b or sustained expression of SUV39H1 delays the repair of HC DNA and reduces clonogenic survival after ionizing irradiation. The results suggest that by regulating JMJD2b and SUV39H1 expression, p53 not only controls transcription but also promotes HC relaxation to accelerate a rate-limiting step in the repair of complex genomes.

Covalent modification of histones has an important role in regulating chromatin dynamics and transcription. Whereas most covalent histone modifications are reversible, until recently it was unknown whether methyl groups could be actively removed from histones. Using a biochemical assay coupled with chromatography, we have purified a novel JmjC domain-containing protein, JHDM1 (JmjC domain-containing histone demethylase 1), that specifically demethylates histone H3 at lysine 36 (H3-K36). In the presence of Fe( ii ) and α-ketoglutarate, JHDM1 demethylates H3-methyl-K36 and generates formaldehyde and succinate. Overexpression of JHDM1 reduced the level of dimethyl-H3-K36 (H3K36me2) in vivo . The demethylase activity of the JmjC domain-containing proteins is conserved, as a JHDM1 homologue in Saccharomyces cerevisiae also has H3-K36 demethylase activity. Thus, we identify the JmjC domain as a novel demethylase signature motif and uncover a protein demethylation mechanism that is conserved from yeast to human.

Histone demethylases, enzymes responsible for removing methyl groups from histone proteins, have emerged as critical players in regulating gene expression and chromatin dynamics, thereby influencing various cellular processes. LSD2 and LSD1 have attracted considerable interest among these demethylases because of their associations with cancer. However, while LSD1 has received significant attention, LSD2 has not been recognized to the same extent. In this study, we conduct a comprehensive comparison between LSD2 and LSD1, with a focus on exploring LSD2’s implications. While both share structural similarities, LSD2 possesses unique features as well. Functionally, LSD2 shows diverse roles, particularly in cancer, with tissue-dependent roles. Additionally, LSD2 extends beyond histone demethylation, impacting DNA methylation, cancer cell reprogramming, E3 ubiquitin ligase activity and DNA damage repair pathways. This study underscores the distinct roles of LSD2, providing insights into their contributions to cancer and other cellular processes.

Up to the mark Two papers in this issue identify enzymes capable of demethylating a trimethyl group from the Lys 9 residue of histone H3 — a 'mark' required for the establishment of heterochromatin and previously considered stable. Cloos et al . show that GASC1, a member of the JMJD2 enzyme family, can disrupt heterochromatin structure when overexpressed and may contribute to tumour development. Klose et al . show that overexpression of JHDM3A, also a JMJD2-type enzyme, disrupts heterochromatin structure. It may function in euchromatin to regulate transcription. One of two papers in this issue that identifies enzymes capable of demethylating a tri-methyl group from Lys 9 of histone H3 — a mark required for the establishment of heterochromatin and previously considered to be stable. GASC1, a member of the JMJD2 enzyme family, can disrupt heterochromatin structure when overexpressed and may contribute to tumour development. Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription 1 , 2 , 3 . Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species 4 , 5 , 6 . H3K9me3 has long been regarded as a ‘permanent’ epigenetic mark 7 , 8 . In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1) 9 , which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C 10 . Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and α-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo . Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas 9 , 11 , 12 , and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.