Histone H3 Lys27 Trimethylation Research Articles

The H1/H5 domain contributes to OsTRBF2 phase separation and gene repression during rice development.

Transcription factors (TFs) tightly control plant development by regulating gene expression. The phase separation of TFs plays a vital role in gene regulation. Many plant TFs have the potential to form phase-separated protein condensates; however, little is known about which TFs are regulated by phase separation and how it affects their roles in plant development. Here, we report that the rice (Oryza sativa) single Myb TF TELOMERE REPEAT-BINDING FACTOR 2 (TRBF2) is highly expressed in fast-growing tissues at the seedling stage. TRBF2 is a transcriptional repressor that binds to the transcriptional start site of thousands of genes. Mutation of TRBF2 leads to pleiotropic developmental defects and misexpression of many genes. TRBF2 displays characteristics consistent with phase separation in vivo and forms phase-separated condensates in vitro. The H1/H5 domain of TRBF2 plays a crucial role in phase separation, chromatin targeting and gene repression. Replacing the H1/H5 domain by a phase-separated intrinsically disordered region from Arabidopsis (Arabidopsis thaliana) AtSERRATE partially recovers the function of TRBF2 in gene repression in vitro and in transgenic plants. We also found that TRBF2 is required for trimethylation of histone H3 Lys27 (H3K27me3) deposition at specific genes and genome-wide. Our findings reveal that phase separation of TRBF2 facilitates gene repression in rice development.

The H1/H5 domain contributes to OsTRBF2 phase separation and gene repression during rice development.

Transcription factors (TFs) tightly control plant development by regulating gene expression. The phase separation of TFs plays a vital role in gene regulation. Many plant TFs have the potential to form phase-separated protein condensates; however, little is known about which TFs are regulated by phase separation and how it affects their roles in plant development. Here, we report that the rice (Oryza sativa) single Myb TF TELOMERE REPEAT-BINDING FACTOR 2 (TRBF2) is highly expressed in fast-growing tissues at the seedling stage. TRBF2 is a transcriptional repressor that binds to the transcriptional start site of thousands of genes. Mutation of TRBF2 leads to pleiotropic developmental defects and misexpression of many genes. TRBF2 displays characteristics consistent with phase separation in vivo and forms phase-separated condensates in vitro. The H1/H5 domain of TRBF2 plays a crucial role in phase separation, chromatin targeting and gene repression. Replacing the H1/H5 domain by a phase-separated intrinsically disordered region from Arabidopsis (Arabidopsis thaliana) AtSERRATE partially recovers the function of TRBF2 in gene repression in vitro and in transgenic plants. We also found that TRBF2 is required for trimethylation of histone H3 Lys27 (H3K27me3) deposition at specific genes and genome-wide. Our findings reveal that phase separation of TRBF2 facilitates gene repression in rice development.

Histone H3.3 chaperone HIRA renders stress-responsive genes poised for prospective lethal stresses in acquired tolerance.

Appropriate responses to environmental challenges are imperative for the survival of all living organisms. Exposure to low-dose stresses is recognized to yield increased cellular fitness, a phenomenon termed hormesis. However, our molecular understanding of how cells respond to low-dose stress remains profoundly limited. Here we report that histone variant H3.3-specific chaperone, HIRA, is required for acquired tolerance, where low-dose heat stress exposure confers resistance to subsequent lethal heat stress. We found that human HIRA activates stress-responsive genes, including HSP70, by depositing histone H3.3 following low-dose stresses. These genes are also marked with histone H3 Lys-4 trimethylation and H3 Lys-9 acetylation, both active chromatin markers. Moreover, depletion of HIRA greatly diminished acquired tolerance, both in normal diploid fibroblasts and in HeLa cells. Collectively, our study revealed that HIRA is required for eliciting adaptive stress responses under environmental fluctuations and is a master regulator of stress tolerance.

Mode of SUV420H2 heterochromatin localization through multiple HP1 binding motifs in the heterochromatic targeting module.

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

Phosphorylated HP1α-Nucleosome Interactions in Phase Separated Environments.

In the nucleus, transcriptionally silent genes are sequestered into heterochromatin compartments comprising nucleosomes decorated with histone H3 Lys9 trimethylation and a protein called HP1α. This protein can form liquid-liquid droplets in vitro and potentially organize heterochromatin through a phase separation mechanism that is promoted by phosphorylation. Elucidating the molecular interactions that drive HP1α phase separation and its consequences on nucleosome structure and dynamics has been challenging due to the viscous and heterogeneous nature of such assemblies. Here, we tackle this problem by a combination of solution and solid-state NMR spectroscopy, which allows us to dissect the interactions of phosphorylated HP1α with nucleosomes in the context of phase separation. Our experiments indicate that phosphorylated human HP1α does not cause any major rearrangements to the nucleosome core, in contrast to the yeast homologue Swi6. Instead, HP1α interacts specifically with the methylated H3 tails and slows the dynamics of the H4 tails. Our results shed light on how phosphorylated HP1α proteins may regulate the heterochromatin landscape, while our approach provides an atomic resolution view of a heterogeneous and dynamic biological system regulated by a complex network of interactions and post-translational modifications.

Epigenetic Regulation of IL-23 by E3 Ligase FBXW7 in Dendritic Cells Is Critical for Psoriasis-like Inflammation.

Dendritic cells (DCs), a driver of psoriasis pathogenesis, produce IL-23 and trigger IL-23/IL-17 cytokine axis activation. However, the mechanisms regulating IL-23 induction remain unclear. In the current study, we found that mice with E3 ligase FBXW7 deficiency in DCs show reduced skin inflammation correlated with the reduction of IL-23/IL-17 axis cytokines in the imiquimod-induced psoriasis model. Fbxw7 deficiency results in decreased production of IL-23 in DCs. FBXW7 interacts with the lysine N-methyltransferase suppressor of variegation 39 homolog 2 (SUV39H2), which catalyzes the trimethylation of histone H3 Lys9 (H3K9) during transcription regulation. FBXW7 mediates the ubiquitination and degradation of SUV39H2, thus decreasing H3K9m3 deposition on the Il23a promoter. The Suv39h2 knockout mice displayed exacerbated skin inflammation with the IL-23/IL-17 axis overactivating in the psoriasis model. Taken together, our results indicate that FBXW7 increases IL-23 expression in DCs by degrading SUV39H2, thereby aggravating psoriasis-like inflammation. Inhibition of FBXW7 or the FBXW7/SUV39H2/IL-23 axis may represent a novel therapeutic approach to psoriasis.

Depletion of enhancer zeste homolog 2 (EZH2) directs transcription factors associated with T cell differentiation through epigenetic regulation of Yin Yang 1(YY1) in combating non-small cell lung cancer (NSCLC).

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of death in all countries alike. In the current study, we have found out that Histone H3Lys4trimethylation is abnormal on YY1 in CD4+T Helper (TH) cells of NSCLC patients which is evident by Histone H3Lys27 trimethylation mediated via EZH2. We investigated the status of Yin Yang 1 (YY1) and the involvement of certain transcription factors that lead to tumorigenesis after depleting endogenous EZH2 in vitro by CRISPR/Cas9 in the CD4+TH1-or-TH2-polarized cells isolated initially as CD4+TH0 cells from the PBMC of the control subjects and patients suffering from NSCLC. After depletion of endogenous EZH2, RT-qPCR based mRNA expression analysis showed that there was an increase in the expression of TH1 specific genes and a decrease in the expression of TH2 specific genes in NSCLC patients CD4+TH cells. We can conclude that this group of NSCLC patients may have the tendency at least in vitro to elucidate adaptive/protective immunity through the depletion of endogenous EZH2 along with the reduction in the expression of YY1. Moreover, depletion of EZH2 not only suppressed the CD4+CD25+FOXP3+Regulatory T cells (Treg) but also it aided the generation of CD8+Cytotoxic T Lymphocytes (CTL) which were involved in killing of the NSCLC cells. Thus the transcription factors involved in EZH2 mediated T cell differentiation linked to malignancies offers us an appealing avenue of targeted therapeutic intervention for NSCLC.

The wheat TaF-box3, SCF ubiquitin ligase component, participates in the regulation of flowering time in transgenic Arabidopsis

Histone methylation is actively involved in plant flowering time and is regulated by a myriad of genetic pathways that integrate endogenous and exogenous signals. We identified an F-box gene from wheat (Triticum aestivum L.) and named it TaF-box3. Transcript expression analysis showed that TaF-box3 expression was gradually induced during the floret development and anthesis stages (WS2.5–10). Furthermore, ubiquitination assays have shown that TaF-box3 is a key component of the SCF ubiquitin ligase complex. TaF-box3 overexpression in Arabidopsis resulted in an early flowering phenotype and different cell sizes in leaves compared to the WT. Furthermore, the transcript level of a flowering time-related gene was significantly reduced in TaF-box3 overexpressing plants, which was linked with lower histone H3 Lys4 trimethylation (H3K4me3) and H3 Lys36 trimethylation (H3K36me3). Overexpression of TaF-box3 in Arabidopsis was shown to be involved in the regulation of flowering time by demethylating FLC chromatin, according to ChIP experiments. Protein analysis confirmed that TaMETS interacts with TaF-box3 and is ubiquitinated and degraded in a TaF-box3-dependnent manner. Based on these findings, we propose that TaF-box3 has a positive role in flowering time, which leads to a better understanding of TaF-box3 physiological mechanism in Arabidopsis.

Targeted Degradation of PRC1 Components, BMI1 and RING1B, via a Novel Protein Complex Degrader Strategy.

Polycomb repressive complex 1 (PRC1) is an essential epigenetic regulator that mainly controls histone H2A Lys119 mono-ubiquitination (H2AK119ub). B cell-specific Moloney murine leukemia virus Integration site 1 (BMI1) and really interesting new gene 1B (RING1B) are PRC1 core components and play critical roles in the development of various cancers. However, therapeutic agents targeting PRC1 are very limited. In this study, MS147, the first degrader of PRC1 core components, BMI1 and RING1B, is discovered via a novel protein complex degradation strategy that utilizes the target protein's interacting partner protein (embryonic ectoderm development (EED)). MS147, which comprises an EED small-molecule binder linked to a ligand of the E3 ligase von Hippel-Lindau (VHL), degrades BMI1/RING1B in an EED-, VHL-, ubiquitination-, and time-dependent manner. MS147 preferentially degrades BMI1/RING1B over polycomb repressive complex 2 (PRC2) core components. Consequently, MS147 effectively reduces H2AK119ub, but not histone H3 Lys27 tri-methylation (H3K27me3), which is catalyzed by PRC2. Furthermore, MS147 effectively inhibits the proliferation of cancer cell lines that are insensitive to PRC2 inhibitors/degraders. Overall, this study provides a novel BMI1/RING1B degrader, which is a useful chemical tool to further investigate the roles of PRC1 in cancer, and a novel protein complex degradation strategy, which can potentially expand the degradable human proteome.

Phytochrome B triggers light-dependent chromatin remodelling through the PRC2-associated PHD finger protein VIL1

To compensate for a sessile nature, plants have developed sophisticated mechanisms to sense varying environmental conditions. Phytochromes (phys) are light and temperature sensors that regulate downstream genes to render plants responsive to environmental stimuli1-4. Here, we show that phyB directly triggers the formation of a repressive chromatin loop by physically interacting with VIN3-LIKE1/VERNALIZATION 5 (VIL1/VRN5), a component of the Polycomb Repressive Complex 2 (PRC2)5, 6, in a light-dependent manner. phyB and VIL1 cooperatively contribute to the repression of growth-promoting genes through the enrichment of Histone H3 Lys 27 trimethylation (H3K27me3), a repressive histone modification. In addition, phyB and VIL1 mediate the formation of a chromatin loop to facilitate the repression of ATHB2. Our findings show that phyB directly utilizes chromatin remodeling to regulate the expression of target genes in a light-dependent manner.

ALKBH5-mediated m6A mRNA methylation governs human embryonic stem cell cardiac commitment

N6-methyladenosine (m6A), as the most abundant modification of mammalian messenger RNAs, is essential for tissue development and pathogenesis. However, the biological significance of m6A methylation in cardiac differentiation and development remains largely unknown. Here, we identify that the downregulation of m6A demethylase ALKBH5 is responsible for the increase of m6A methylation and cardiomyocyte fate determination of human embryonic stem cells (hESCs) from mesoderm cells (MESs). In contrast, ALKBH5 overexpression remarkably blocks cardiomyocyte differentiation of hESCs. Mechanistically, KDM5B and RBBP5, the components of H3K4 modifying enzyme complexes, are identified as downstream targets for ALKBH5 in cardiac-committed hESCs. Loss of function of ALKBH5 alters the expression of KDM5B and RBBP5 through impairing stability of their mRNAs, which in turn promotes the transcription of GATA4 by enhancing histone H3 Lys4 trimethylation (H3K4me3) at the promoter region of GATA4. Taken together, we reveal a previously unidentified role of m6A demethylase ALKBH5 in determining cardiac lineage commitment of hESCs.

Hypoxia-inducible factor-1α cooperates with histone Lys methylation to predict prognosis in esophageal squamous cell carcinoma.

Aim: To investigate hypoxia-inducible factor-1α(HIF-1α) and histone methylation markers as potential indicators of prognosis in esophageal squamous cell carcinoma (ESCC). Patients &methods: The prognostic value of HIF-1α and histone methylation markers levelswas analyzed using Kaplan-Meier survival analysis. Results: HIF-1α protein expression was higher in ESCC tumors than in paracancerous tissues. Histone H3 Lys9 trimethylation (H3K9me3), histone H3 Lys27 trimethylation (H3K27me3), histone H3 Lys4trimethylation (H3K4me3)and histone H4 Lys20 trimethylation (H4K20me3) were significantly upregulated in ESCC tissues. HIF-1α was only positively correlated with H3K9me3 and H3K4me3 expression. Kaplan-Meier analysis indicated that H3K9me3, H3K27me3, H4K20me3 and histone H3 Lys36 trimethylation (H3K36me3) were independent indicators of prognosis for ESCC. Conclusion: This study identified a pattern of epigenetic methylation markers and HIF-1α expression in ESCC, and their combined evaluation might improve survival prediction for ESCC patients.

Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression.

Methods for quantifying gene expression1 and chromatin accessibility2 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 method3 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.

O-linked N-acetylglucosaminyltransferase OGT inhibits diabetic nephropathy by stabilizing histone methyltransferases EZH2 via the HES1/PTEN axis

BackgroundO-linked N-acetylglucosaminyltransferase (OGT) is involved in diabetes-related diseases including diabetic nephropathy (DN), and responsible for O-GlcNAcylation. Moreover, O-GlcNAcylation and OGT could be induced by high glucose. Thus, we sought to explore the molecular mechanism of OGT in DN. MethodsLoss- and gain-functions were conducted to determine the roles of OGT, enhancer of zeste homolog 2 (EZH2), hairy and enhancer of split 1 (HES1) and phosphatase and tensin homolog (PTEN) in the viability, cell cycle and fibrosis of mesangial cells (MCs), followed by the assessment using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blot assay (fibrosis-related proteins). The interaction between OGT and EZH2 and the effect on EZH2 glycosylation were verified by chromatin immunoprecipitation (ChIP) and glutathione S-transferase (GST) pull-down assays. EZH2 stability was checked by treatment with cycloheximide. ResultsExpression of OGT was repressed in the DN mice and high glucose-treated MCs. Elevated OGT suppressed viability of high glucose-treated MCs, blocked proliferation characterized by repressed cyclin D1, but enhanced p21 levels, and inhibited fibrosis evidenced by reduced levels of fibronectin (FN) and collagen-4 (col-4). OGT interacted with EZH2 and promoted its glycosylation thus stabilizing the EZH2. EZH2 overexpression enhanced the enrichment of EZH2 and histone H3 Lys27 trimethylation (H3K27me3) in the HES1 promoter. HES1 was upregulated and PTEN was downregulated in DN mice. Transduction of lentivirus vector containing overexpression (oe)-OGT alleviated renal injury in DN mice. ConclusionsCollectively, OGT stabilizes histone methyltransferases EZH2 to regulate HES1/PTEN thus inhibiting DN.

Dynamic Behavior of Inactive X Chromosome Territory During the Cell Cycle as Revealed by H3K27me3-Specific Intracellular Antibody.

Posttranslational histone modifications are critical for the regulation of genome function. The levels of histone modifications oscillate during the cell cycle. Most modifications are diluted after DNA replication and then their levels are restored during the rest of the cell cycle with different kinetics depending on the modification. Some modifications, like histone H4 Lys20 monomethylation (H4K20me1), exhibit cell cycle-dependent dynamic changes. To track histone modifications in living cells, we have developed genetically encoded probes termed modification specific intracellular antibodies, or "mintbodies." As mintbodies shuttle between the cytoplasm and nucleus by diffusion, their nuclear concentration depends on the target modification level. By measuring the nuclear to cytoplasmic intensity ratio of H4K20me1-specific mintbody, we have monitored the increase of H4K20me1 in the G2 phase. Here we describe how the mintbody-based methods can be applied to track a specific chromosome, such as the inactive X chromosome (Xi), on which genes are repressed through histone H3 Lys27 trimethylation (H3K27me3). When H3K27me3-specific mintbodies are expressed in cells that harbor Xi, the mintbodies are concentrated on Xi and the dynamic behavior of Xi can be tracked using a confocal microscope. After acquiring 3D time-lapse images, an image analysis allows measuring the volume, shape and H3K27me3 level of Xi during the cell cycle.

The transcriptional repressors VAL1 and VAL2 recruit PRC2 for genome-wide Polycomb silencing in Arabidopsis

The Polycomb repressive complex 2 (PRC2) catalyzes histone H3 Lys27 trimethylation (H3K27me3) to repress gene transcription in multicellular eukaryotes. Despite its importance in gene silencing and cellular differentiation, how PRC2 is recruited to target loci is still not fully understood. Here, we report genome-wide evidence for the recruitment of PRC2 by the transcriptional repressors VIVIPAROUS1/ABI3-LIKE1 (VAL1) and VAL2 in Arabidopsis thaliana. We show that the val1 val2 double mutant possesses somatic embryonic phenotypes and a transcriptome strikingly similar to those of the swn clf double mutant, which lacks the PRC2 catalytic subunits SWINGER (SWN) and CURLY LEAF (CLF). We further show that VAL1 and VAL2 physically interact with SWN and CLF in vivo. Genome-wide binding profiling demonstrated that they colocalize with SWN and CLF at PRC2 target loci. Loss of VAL1/2 significantly reduces SWN and CLF enrichment at PRC2 target loci and leads to a genome-wide redistribution of H3K27me3 that strongly affects transcription. Finally, we provide evidence that the VAL1/VAL2–RY regulatory system is largely independent of previously identified modules for Polycomb silencing in plants. Together, our work demonstrates an extensive genome-wide interaction between VAL1/2 and PRC2 and provides mechanistic insights into the establishment of Polycomb silencing in plants.

PRC2 Acts as a Critical Timer That Drives Oligodendrocyte Fate over Astrocyte Identity by Repressing the Notch Pathway

SUMMARYPRC2 creates the repressive mark histone H3 Lys27 trimethylation. Although PRC2 is involved in various biological processes, its role in glial development remains ambiguous. Here, we show that PRC2 is required for oligodendrocyte (OL) differentiation and myelination, but not for OL precursor formation. PRC2-deficient OL lineage cells differentiate into OL precursors, but they fail to trigger the molecular program for myelination, highlighting that PRC2 is essential for directing the differentiation timing of OL precursors. PRC2 null OL lineage cells aberrantly induce Notch pathway genes and acquire astrocytic features. The repression of the Notch pathway restores the myelination program and inhibits abnormal astrocytic differentiation in the PRC2-deficient OL lineage, indicating that Notch is a major target of PRC2. Altogether, our studies propose a specific action of PRC2 as a novel gatekeeper that determines the glial fate choice and the timing of OL lineage progression and myelination by impinging on the Notch pathway.

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

Endothelial progenitor cells-secreted extracellular vesicles containing microRNA-93-5p confer protection against sepsis-induced acute kidney injury via the KDM6B/H3K27me3/TNF-α axis

The pivotal pathogenetic role of microRNAs (miRs) in sepsis-induced acute kidney injury (AKI) has been demonstrated in mounting evidence. The functions of the target cells are regulated through the release of cells-encapsulated extracellular vesicles (Evs) into the extracellular space. The present study aims to elucidate the clinical significance as well as biological function of the endothelial progenitor cell (EPC)-derived Evs containing miR-93-5p in sepsis-induced AKI. We first established a cellular sepsis-induced AKI mouse model by treatment with lipopolysaccharide (LPS), and tested ectopic expression and depletion experiments in the model. Evs derived from miR-93-5p inhibitor-transfected EPCs (Evs/miR-93-5p inhibitor) were isolated, and co-cultured with HK2 cells to explore the effects of EPC-derived Evs overexpressing miR-93-5p on LPS-induced HK2 cell injury. The interaction between miR-93-5p and lysine (K)-specific demethylase 6B (KDM6B) was identified using dual-luciferase reporter assay, and ChIP was used to validate the relationship between KDM6B and tumor necrosis factor-α (TNF-α). Mice were made septic by cecal ligation and puncture (CLP), and then injected with Ev/miR-93-5p inhibitor to explore its functions in vivo. The results found that miR-93-5p and histone H3 Lys27 trimethylation (H3K27me3) were downregulated while KDM6B was upregulated in LPS-treated HK2 cells. EPC-derived Evs alleviated LPS-induced HK2 cell injury, while Ev/miR-93-5p inhibitor potentiated the cell injury in vitro. miR-93-5p was found to directly target KDM6B. Silencing KDM6B induced H3K27me3, inhibiting the activation of TNF-α, thereby weakening LPS-induced HK2 cell injury. EPC-derived Evs containing miR-93-5p attenuated multiple organ injury, vascular leakage, inflammation, and apoptosis in septic mice. In conclusion, the present study demonstrated that endothelial protection from EPC-derived Evs carrying miR-93-5p in sepsis-induced AKI, which was mediated by regulation KDM6BH/3K27me3/TNF-α axis.

Binding and allosteric transmission of histone H3 Lys-4 trimethylation to the recombinase RAG-1 are separable functions of the RAG-2 plant homeodomain finger

V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.